Sources of error in the isotopic cholesterol balance method in African green monkeys consuming a cholesterol-free diet

Six African green monkeys were labeled intravenously with [1,2-(3)H]cholesterol while consuming a cholesterol-free liquid formula diet. The plasma cholesterol specific activity was compared with the specific activity of the biliary cholesterol and bile acids and with the fecal neutral steroids in order to determine whether the traditional isotopic balance method was valid for the calculation of endogenous cholesterol excretion. The specific activity of biliary cholesterol and bile acids averaged 10-15% lower than plasma cholesterol specific activity. Fecal cholesterol and coprostanone specific activities were similar to that of the biliary cholesterol, but the specific activity of fecal coprostanol was approximately 25% lower. This suggests that biliary cholesterol and bile acids were derived from a pool of hepatic cholesterol that did not completely equilibrate with the whole body exchangeable cholesterol pool. In addition, there was further reduction in the specific activity of coprostanol, the major fecal neutral steroid, presumably by cholesterol synthesized in the lower intestine and preferentially converted to coprostanol. As a result, the traditional isotopic balance procedure underestimated endogenous neutral steroid excretion by 46% and bile acid excretion by 31% in African green monkeys fed the cholesterol-free diet. Within 7 days after the addition of 1 mg cholesterol/kcal to the diet, the specific activities of plasma and biliary cholesterol and biliary bile acids were identical and there was no difference in the specific activities of the individual fecal neutral steroids. Thus, the traditional isotopic balance procedure (DPM fecal neutral steroids + bile acids/specific activity [DPM/mg] plasma cholesterol) can be used for calculation of endogenous cholesterol excretion in cholesterol-fed animals during the nonsteady state when plasma cholesterol concentrations are rapidly increasing, as well as after a new steady state has been achieved.-Henderson, G. R., and R. W. St. Clair. Sources of error in the isotopic cholesterol balance method in African green monkeys consuming a cholesterol-free diet.     

Cholesterol gallstones in alloxan-diabetic mice

Normal and alloxan-diabetic male mice (Crj-ICR) were fed a diet containing 0.5% cholesterol for 5 and 10 weeks, and gallbladder bile was analyzed for cholesterol, phospholipids and bile acids, feces for sterols and bile acids, and plasma and liver for cholesterol, phospholipids, and triglycerides. Normal mice developed no gallstones but the diabetic mice developed cholesterol gallstones with an incidence of 70% by 5 weeks and 80% by 10 weeks after feeding of the cholesterol diet. Diabetic mice fed the ordinary diet also developed stones (23%) by 10 weeks. In the diabetic mice, the gallbladder was enlarged about threefold, and biliary lipid concentration, diet intake, and fecal excretion of sterols and bile acids increased but body weight decreased. Cholic acid and beta-muricholic acid comprised over 40% each of the total biliary bile acids in normal mice, but cholic acid increased to about 80% and beta-muricholic acid decreased to a few percent in the diabetic mice. Fecal excretion of bile acids increased after cholesterol feeding in both normal and diabetic mice, but the increased bile acid in the normal animals was beta-muricholic acid and that in the diabetic mice was deoxycholic acid. The mice that developed gallstones showed a marked increase in biliary cholesterol value and decreases in gallbladder bile and bile acid concentration, but no difference in biliary and fecal bile acid composition, bile acid synthesis, fecal sterols, or plasma and liver lipid levels. Cholesterol absorption was increased in the diabetic mice when examined by plasma 14C/3H ratio and fecal 14C-labeled sterol excretion after a single oral administration of [14C]cholesterol and a simultaneous intravenous injection of [3H]cholesterol. These data led to the conclusion that cholesterol gallstones developed in alloxan-diabetic mice fed excess cholesterol, due to the hyperphagia and the enhancement of cholesterol absorption caused by increases in the synthesis and secretion of cholic acid.     

Inhibition of cholesterol absorption by SCH 58053 in the mouse is not mediated via changes in the expression of mRNA for ABCA1, ABCG5, or ABCG8 in the enterocyte

Intestinal cholesterol absorption is a major determinant of plasma low density lipoprotein-cholesterol (LDL-C) concentrations. Ezetimibe (SCH 58235) and its analogs SCH 48461 and SCH 58053 are novel potent inhibitors of cholesterol absorption whose mechanism of action is unknown. These studies investigated the effect of SCH 58053 on cholesterol metabolism in female 129/Sv mice. In mice fed a low cholesterol rodent diet containing SCH 58053, cholesterol absorption was reduced by 46% and fecal neutral sterol excretion was increased 67%, but biliary lipid composition and bile acid synthesis, pool size, and pool composition were unchanged. When the dietary cholesterol content was increased either 10- or 50-fold, those animals given SCH 58053 manifested lower hepatic and biliary cholesterol concentrations than did their untreated controls. Cholesterol feeding increased the relative mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1), ABC transporter G5 (ABCG5), and ABC transporter G8 (ABCG8) in the jejunum, and of ABCG5 and ABCG8 in the liver, but the magnitude of this increase was generally less if the mice were given SCH 58053. We conclude that the inhibition of cholesterol absorption effected by this new class of agents is not mediated via changes in either the size or composition of the intestinal bile acid pool, or the level of mRNA expression of proteins that facilitate cholesterol efflux from the enterocyte, but rather may involve disruption of the uptake of luminal sterol across the microvillus membrane.     

Hepatic cholesterol and bile acid metabolism and intestinal cholesterol absorption in scavenger receptor class B type I-deficient mice

The scavenger receptor class B type I (SR-BI), which is expressed in the liver and intestine, plays a critical role in cholesterol metabolism in rodents. While hepatic SR-BI expression controls high density lipoprotein (HDL) cholesterol metabolism, intestinal SR-BI has been proposed to facilitate cholesterol absorption. To evaluate further the relevance of SR-BI in the enterohepatic circulation of cholesterol and bile salts, we studied biliary lipid secretion, hepatic sterol content and synthesis, bile acid metabolism, fecal neutral sterol excretion, and intestinal cholesterol absorption in SR-BI knockout mice. SR-BI deficiency selectively impaired biliary cholesterol secretion, without concomitant changes in either biliary bile acid or phospholipid secretion. Hepatic total and unesterified cholesterol contents were slightly increased in SR-BI-deficient mice, while sterol synthesis was not significantly changed. Bile acid pool size and composition, as well as fecal bile acid excretion, were not altered in SR-BI knockout mice. Intestinal cholesterol absorption was somewhat increased and fecal sterol excretion was slightly decreased in SR-BI knockout mice relative to controls. These findings establish the critical role of hepatic SR-BI expression in selectively controlling the utilization of HDL cholesterol for biliary secretion. In contrast, SR-BI expression is not essential for intestinal cholesterol absorption.     

Delineation of molecular changes in intrahepatic cholesterol metabolism resulting from diminished cholesterol absorption

The absorption of cholesterol by the small intestine is a major route for the net entry of cholesterol into the body and can therefore affect the plasma low density lipoprotein-cholesterol (LDL-C) concentration. These studies used ezetimibe, a potent inhibitor of cholesterol absorption, to delineate the biochemical and molecular changes in intrahepatic metabolism and biliary lipid secretion when there is a major reduction in chylomicron cholesterol delivery to the liver. In female LDL receptor (LDLR)-deficient (LDLR-/-) mice fed a basal diet containing ezetimibe (0-10 mg/day/kg body weight), cholesterol absorption was reduced up to 91%, fecal neutral sterol excretion was increased up to 4.7-fold, and plasma total cholesterol concentrations decreased by up to 18%. Blocking cholesterol absorption prevented the accumulation of very low density lipoproteins and LDL in the circulation of LDLR-/- mice fed a lipid-rich diet. In female LDLR+/+ mice fed the lipid-rich diet with ezetimibe, the relative mRNA level for the LDLR in the liver was 2-fold greater than in matching mice given the lipid-rich diet alone. We conclude that in the mouse the reduction in plasma LDL-C levels induced by blocking cholesterol absorption reflects both a diminished rate of LDL-C production and a modest increase in hepatic LDLR expression.     

Disruption of the sterol carrier protein 2 gene in mice impairs biliary lipid and hepatic cholesterol metabolism.

Hepatic up-regulation of sterol carrier protein 2 (Scp2) in mice promotes hypersecretion of cholesterol into bile and gallstone formation in response to a lithogenic diet. We hypothesized that Scp2 deficiency may alter biliary lipid secretion and hepatic cholesterol metabolism. Male gallstone-susceptible C57BL/6 and C57BL/6(Scp2(-/-)) knockout mice were fed a standard chow or lithogenic diet. Hepatic biles were collected to determine biliary lipid secretion rates, bile flow, and bile salt pool size. Plasma lipoprotein distribution was investigated, and gene expression of cytosolic lipid-binding proteins, lipoprotein receptors, hepatic regulatory enzymes, and intestinal cholesterol absorption was measured. Compared with chow-fed wild-type animals, C57BL/6(Scp2(-/-)) mice had higher bile flow and lower bile salt secretion rates, decreased hepatic apolipoprotein expression, increased hepatic cholesterol synthesis, and up-regulation of liver fatty acid-binding protein. In addition, the bile salt pool size was reduced and intestinal cholesterol absorption was unaltered in C57BL/6(Scp2(-/-)) mice. When C57BL/6(Scp2(-/-)) mice were challenged with a lithogenic diet, a smaller increase of hepatic free cholesterol failed to suppress cholesterol synthesis and biliary cholesterol secretion increased to a much smaller extent than phospholipid and bile salt secretion. Scp2 deficiency did not prevent gallstone formation and may be compensated in part by hepatic up-regulation of liver fatty acid-binding protein. These results support a role of Scp2 in hepatic cholesterol metabolism, biliary lipid secretion, and intracellular cholesterol distribution.     

Effect of ethanol on cholesterol and bile acid metabolism

Ethanol feeding increased significantly levels of hepatic esterified cholesterol and serum free and esterified cholesterol in rats. Incorporation of intraperitoneally administered [(14)C]acetate into cholesterol was significantly increased. Labeling of cholesterol was also enhanced in liver slices from animals pretreated with ethanol and incubated with [(14)C]-acetate. Ethanol consumption prolonged the half-excretion time of labeled cholic or chenodeoxycholic acids, increased slightly the pool size, and decreased daily excretion. By contrast, supplementation of the diet with cholesterol shortened the half-excretion time, did not modify pool size, and increased daily excretion. When ethanol and cholesterol feeding were combined, the effects of ethanol prevailed and there was suppression of the adaptive changes in bile acid metabolism induced by cholesterol feeding. There was also a greater accumulation of esterified cholesterol in the liver than that produced by cholesterol alone, ethanol administration alone, or the summation of both effects. Thus, cholesterol accumulation produced by ethanol feeding is associated with both enhanced cholesterogenesis and decreased bile acid excretion. Both mechanisms may play a role, but the latter is probably predominant in these studies in which cholesterol accumulation was markedly enhanced by the addition of cholesterol to the ethanol-containing diet.     

Contraceptive steroids increase cholesterol in bile: mechanisms of action

Contraceptive steroids increase the risk of acquiring cholesterol gallstones. The factors responsible include an increase in cholesterol saturation of bile and an increase in rate of secretion of cholesterol into bile. The goal of this study was to investigate the mechanism(s) of these increases in biliary cholesterol. During the use of contraceptive steroids, cholesterol saturation of gallbladder bile and the amount of cholesterol secreted per mole of bile acid increased (P less than 0.05 and P less than 0.02, respectively). Cholesterol absorption, cholesterol synthesis, chylomicron remnant clearance, and the concentration of plasma and lipoprotein lipids were not altered by contraceptive steroids. Despite this apparent lack of effect, important correlations were present during steroid use. LDL (low density lipoprotein) cholesterol increased as dietary cholesterol increased (r = 0.58, P less than 0.025). Cholesterol synthesis correlated directly with VLDL cholesterol concentration (r = 0.64, P less than 0.01), biliary cholesterol secretion (r = 0.68, P less than 0.01) and with molar percent cholesterol in bile (r = 0.49, P = 0.06). Chylomicron remnant clearance also correlated with cholesterol secretion (r = 0.85, P less than 0.001). As either remnant uptake or synthesis increased, the effect of the other source of hepatic cholesterol on biliary cholesterol secretion diminished. These relationships were not observed in the same subjects when they were not taking the hormones. The findings suggest that both newly synthesized and dietary cholesterol contribute to the cholesterol secreted in bile. This is consistent with the hypothesis that cholesterol for secretion into bile and VLDL is derived from a common metabolic pool of free cholesterol. It is proposed that contraceptive steroids exert their effect on biliary cholesterol by increasing cholesterol entering the pool and/or by inhibiting hepatic ACAT (acylcoenzyme A:cholesterol acyltransferase) activity, a known effect of progesterone, so that an increase in free cholesterol entering the pool leads to an increase in output.     

Physical exercise modifies the effect of high cholesterol-sucrose feeding in the rat.

1. Adult male rats were fed a basic chow (less than 0.01% cholesterol) and the same diet modified to contain 0.2% cholesterol and 20% sucrose. 2. Cholesterol-sucrose diet increased the erythrocyte cholesterol and the liver cholesterol. This diet decreased the epididymal fat weight and the biliary cholesterol and it improved the micellar solubility of cholesterol in the bile. 3. Swimming daily for 1 h for 94 days modified the effect of cholesterol-sucrose feeding: it induced plasma lecithin-cholesterol acyltransferase (LCAT), it decreased erythrocyte cholesterol, plasma total and unesterified cholesterol, and adipose tissue phospholipids to a level even beyond that of the animals on the basic chow. These changes in lipid levels induced by exercise suggest an important role of LCAT in cholesterol transport. 4. Exercise did not effect micellar solubility of cholesterol in the bile probably because cholesterol biosynthesis was already suppressed by dietary cholesterol. 5. Exercise promotes cholesterol esterification and transport from the peripheral tissues to the liver not only on a low cholesterol diet (our previous reports) but also when feeding a diet high in cholesterol and sucrose.     

The origin of cholesterol in the mesenteric lymph of the rat

These studies were performed to quantitate the amounts of newly synthesized cholesterol secreted in the mesenteric lymph of the rat and to define the origin of this cholesterol. In control animals receiving no dietary fat, the amount of newly synthesized sterol entering the lymph increased linearly with respect to time over 24 hr. When a continuous intravenous infusion of chylomicrons was given or when the animals were prefed a diet containing 2.0% cholesterol to inhibit hepatic, but not intestinal or peripheral, cholesterol synthesis, the secretion of newly synthesized sterol in lymph was markedly suppressed, suggesting that the liver was its ultimate site of origin. When the animals were subjected to either blockade of intestinal cholesterol absorption or biliary diversion, there was a decrease in both the newly synthesized and total mass of cholesterol in lymph by approximately 60%, indicating that the majority was normally derived from the absorption of luminal (primarily biliary) sterol. In the absence of dietary cholesterol, the remainder was probably derived from plasma lipoproteins that were filtered through the intestinal capillaries into the lymph. In contrast, when lymph was collected during active fat absorption, the intestine was found to secrete sterol newly synthesized by the epithelium. Such newly synthesized cholesterol was found predominantly in the unesterified fraction and accounted for approximately 27% of the total sterol found in lymph at the end of the experiment. From these studies it was concluded that in the absence of fat absorption, sterol synthesized in the intestinal mucosa was incorporated predominantly into cell membranes and did not enter intestinal lymph to any significant degree. However, during fat absorption, a fraction of this newly synthesized sterol pool was incorporated into lipoproteins and so was delivered through the intestinal lymph to the body pools of cholesterol.     

Mechanisms of action of clofibrate on cholesterol metabolism in patients with hyperlipidemia

The influence of clofibrate on cholesterol metabolism in patients with hyperlipidemia was studied by means of sterol balance and isotope kinetic techniques and by measurements of flow rates of cholesterol through the biliary tract. Long-term balance studies were carried out on a metabolic ward in 24 patients with all currently recognized types of hyperlipidemia; in five other patients with hypercholesterolemia, pool sizes and turnover rates of cholesterol were defined by compartmental analysis before and after three years' daily administration of the drug. Except in fat-induced hypertriglyceridemia (two patients), clofibrate caused reduced plasma levels of triglycerides and cholesterol in all categories of hyperlipidemia. As a general rule, excretion of cholesterol into bile and feces was significantly increased and fecal bile acid excretion was decreased, regardless of the type of lipoprotein abnormality. Despite a net increase in steroid excretion in most patients with hyperlipidemia, cholesterol synthesis was not increased; indeed, in many patients synthesis appeared to be decreased. While the data obtained in 29 patients were not always consistent, the bulk of the evidence suggests that, in all forms of hyperlipidemia except fat-induced hyperglyceridemia, the drug causes an increased output of cholesterol while simultaneously inhibiting any compensatory increase in cholesterol synthesis. Therefore, it appeared that the increased excretion of steroids was most likely derived from cholesterol stored in tissues. This conclusion was strengthened by finding that long-term administration of the drug can cause marked reduction in body pools of cholesterol. These findings are reflected clinically by resolution of skin and tendon xanthomatosis. However, it is not yet known whether the accumulation of cholesterol in arterial walls that is part of the process of atherogenesis can be inhibited or reversed by the drug.     

Enrichment of canalicular membrane with cholesterol and sphingomyelin prevents bile salt-induced hepatic damage

These studies were undertaken to characterize the role of plasma membrane cholesterol in canalicular secretory functions and hepatocyte integrity against intravenous taurocholate administration. Cholesterol and sphingomyelin concentrations and cholesterol/phospholipid ratios were significantly increased in canalicular membranes of diosgenin-fed rats, suggesting a more resistant structure against solubilization by taurocholate. During taurocholate infusion, control rats had significantly decreased bile flow, whereas diosgenin-fed animals maintained bile flow. Maximal cholesterol output increased by 176% in diosgenin-fed rats, suggesting an increased precursor pool of biliary cholesterol in these animals. Maximal phospholipid output only increased by 43% in diosgenin-fed rats, whereas bile salt output remained at control levels. The kinetics of glutamic oxalacetic transaminase, lactic dehydrogenase, and alkaline phosphatase activities in bile showed a significantly faster release in control than in diosgenin-fed rats. After 30 min of intravenous taurocholate infusion, necrotic hepatocytes were significantly increased in control animals. Preservation of bile secretory functions and hepatocellular cytoprotection by diosgenin against the intravenous infusion of toxic doses of taurocholate was associated with an increased concentration of cholesterol and sphingomyelin in the canalicular membrane. The increase of biliary cholesterol output induced by diosgenin was correlated to the enhanced concentration of cholesterol in the canalicular membrane.     

Effects of plasma cholesterol lowering agents on hepatobiliary lipid metabolism and cholesterol turnover in the rhesus monkey.

The effects of clofibrate, cholestyramine, and neomycin on hepatobiliary lipid metabolism were studied in adult rhesus monkeys in metabolic steady state with intact but exteriorized enterohepatic circulations. Clofibrate (30 mg/kg, id) had no effect on lipid secretion while cholestyramine (150 mg/kg, id) decreased biliary cholesterol secretion rate from 0.19 +/- 0.03 to 0.13 +/- 0.02 mmol/24 h, p less than 0.05. Neomycin (30 mg/kg, id) decreased bile flow from 216 +/- 10 to 191 +/- 7mL/24 h, p less than 0.05, and tended only to decrease bile salt and phospholipid secretion rates. Cholestyramine decreased cholesterol composition from 1.81 +/- 0.22 to 1.30 +/- 0.22 mol %, p less than 0.05, while clofibrate and neomycin had insignificant effects. Cholestyramine and neomycin decreased bile salt pool size from 1 +/- 0.1 to 0.77 +/- 0.15 and from 1.45 +/- 0.16 to 1.13 +/- 0.21 mmol, p less than 0.05, respectively, while clofibrate had no effect. Bile salt synthetic rate was increased only by cholestyramine, i.e., from 0.63 +/- 0.04 to 1.48 +/- 0.26 mmol/24 h, p less than 0.01. Concomitant cholesterol turnover studies revealed that cholestyramine increased the production rate and excretion of cholesterol in the rapidly miscible cholesterol pool and increased the transfer of cholesterol from slow to rapidly miscible pools. Neomycin, on the other hand, decreased the size of the rapidly miscible pool by decreasing production rate without affecting the size of the slowly miscible pool, while clofibrate had insignificant effects.     

The influence of estrogen on hepatic cholesterol metabolism and biliary lipid secretion in rats fed fish oil

Both estrogen and dietary n-3 polyunsaturated fatty acids are known to be hypocholesterolemic, but appear to exert their effects by different mechanisms. In this study, the interaction between dietary fish oil (rich in n-3 polyunsaturated fatty acids) and estrogen in the regulation of hepatic cholesterol metabolism and biliary lipid secretion in rats was studied. Rats fed a low fat or a fish oil-supplemented diet for 21 days were injected with 17alpha-ethinyl estradiol (5 mg/kg body weight) or the vehicle only (control rats) once per day for 3 consecutive days. Estrogen-treatment led to a marked reduction in plasma cholesterol levels in fish oil-fed rats, which was greater than that observed with either estrogen or dietary fish oil alone. The expression of mRNA for cholesterol 7alpha-hydroxylase was decreased by estrogen in rats fed a low fat or a fish oil-supplemented diet, while the output of cholesterol (micromol/h/kg b.wt.) in the bile was unchanged in both groups. Cholesterol levels in the liver were increased by estrogen in rats given either diet, but there was a significant shift from cholesterol esterification to cholesteryl ester hydrolysis only in the fish oil-fed animals. Estrogen increased the concentration of cholesterol (micromol/ml) in the bile in rats fed the fish oil, but not the low fat diet. However, the cholesterol saturation index was unaffected. The output and concentration of total bile acid was also unaffected, but changes in the distribution of the individual bile acids were observed with estrogen treatment in both low fat and fish oil-fed groups. These results show that interaction between estrogen-treatment and dietary n-3 polyunsaturated fatty acids causes changes in hepatic cholesterol metabolism and biliary lipid secretion in rats, but does not increase the excretion of cholesterol from the body.     

Inhibiting intestinal NPC1L1 activity prevents diet-induced increase in biliary cholesterol in Golden Syrian hamsters

Niemann-Pick C1-like 1 (NPC1L1) facilitates the uptake of sterols into the enterocyte and is the target of the novel cholesterol absorption inhibitor, ezetimibe. These studies used the Golden Syrian hamster as a model to delineate the changes in the relative mRNA expression of NPC1L1 and other proteins that regulate sterol homeostasis in the enterocyte during and following cessation of ezetimibe treatment and also to address the clinically important question of whether the marked inhibition of cholesterol absorption alters biliary lipid composition. In hamsters fed a low-cholesterol, low-fat basal diet, the abundance of mRNA for NPC1L1 in the small intestine far exceeded that in other regions of the gastrointestinal tract, liver, and gallbladder. In the first study, female hamsters were fed the basal diet containing ezetimibe at doses up to 2.0 mg.day(-1).kg body wt(-1). At this dose, cholesterol absorption fell by 82%, fecal neutral sterol excretion increased by 5.3-fold, and hepatic and intestinal cholesterol synthesis increased more than twofold, but there were no significant changes in either fecal bile acid excretion or biliary lipid composition. The ezetimibe-induced changes in intestinal cholesterol handling were reversed when treatment was withdrawn. In a second study, male hamsters were given a diet enriched in cholesterol and safflower oil without or with ezetimibe. The lipid-rich diet raised the absolute and relative cholesterol levels in bile more than fourfold. This increase was largely prevented by ezetimibe. These data are consistent with the recent finding that ezetimibe treatment significantly reduced biliary cholesterol saturation in patients with gallstones.     

Effects of dietary soybean lecithin on plasma lipid transport and hepatic cholesterol metabolism in rats

Dietary lecithin can stimulate bile formation and biliary lipid secretion, particularly cholesterol output in bile. Studies also suggested that the lecithin-rich diet might modify hepatic cholesterol homeostasis and lipoprotein metabolism. Therefore, we examined hepatic activities of 3-hydroxy-3 methylglutaryl coenzyme A reductase "HMG -CoA reductase", cholesterol 7 alpha-hydroxylase and acyl-CoA: cholesterol acyltransferase "ACAT" as well as plasma lipids and lipoprotein composition in rats fed diets enriched with 20% of soybean lecithin during 14 days. We also evaluated the content of hepatic canalicular membrane proteins involved in lipid transport to the bile (all P-glycoproteins as detected by the C 219 antibody and the sister of P-glycoprotein "spgp" or bile acid export pump) by Western blotting. As predicted, lecithin diet modified hepatic cholesterol homeostasis. The activity of hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was enhanced by 30 and 12% respectively, while microsomal ACAT activity showed a dramatic decrease of 75%. As previously reported from ACAT inhibition, the plasma level and size of very low-density lipoprotein (VLDL) were significantly decreased and bile acid pool size and biliary lipid output were significantly increased. The canalicular membrane content of lipid transporters was not significantly affected by dietary lecithin. The current data on inhibition of ACAT activity and related metabolic effects by lecithin mimic the previously reported effects following drug-induced inhibition of ACAT activity, suggesting potential beneficial effects of dietary lecithin supplementation in vascular disease.     

Effect of bean intake on biliary lipid secretion and on hepatic cholesterol metabolism in the rat

We studied the effect of a bean diet on biliary lipid secretion, serum cholesterol concentration, and hepatic cholesterol metabolism in the rat. Rats fed a bean diet for 10-12 days had increased biliary cholesterol output and molar percentage by 300% and 200%, respectively, compared to rats fed an isocaloric and isoprotein casein diet. Biliary phospholipid output increased 180%. Bile flow and biliary bile salt output remained in the normal range. Total serum and VLDL cholesterol concentration significantly decreased 27% and 50%, respectively, in the rats fed the bean diet. Hepatic cholesterogenesis was increased 170% in the bean-fed animals. The relative contribution of newly synthesized hepatic cholesterol to total biliary cholesterol increased 200%, and that of endogenous origin only 50%. These results suggested that newly synthesized hepatic cholesterol was preferentially channelled to the biliary cholesterol secretory pathway in bean-fed rats. Although hepatic cholesteryl ester concentration increased 240%, the incorporation of [14C]oleate into hepatic cholesteryl esters was significantly decreased by 30% in isolated hepatocytes of bean-fed animals. These results were consistent with the possibility that the availability of hepatic free cholesterol for biliary secretion was increased in the bean-fed animals. This study demonstrates that bean intake has a profound effect on the metabolic channelling and compartmentalization of hepatic cholesterol, resulting in a significant decrease in total serum and very low density lipoprotein cholesterol concentrations and a high biliary cholesterol output.     

Comparison of synthetic saponin cholesterol absorption inhibitors in rabbits: evidence for a non-stoichiometric, intestinal mechanism of action

The hypocholesterolemic activities of pamaqueside and tiqueside, two structurally similar saponins, were evaluated in cholesterol-fed rabbits. The pharmacological profiles of the saponins were virtually identical: both dose-dependently decreased the intestinal absorption of labeled cholesterol 25-75%, increased fecal neutral sterol excretion up to 2.5-fold, and decreased hepatic cholesterol content 10-55%. High doses of pamaqueside (>5 mg/kg) or tiqueside (>125 mg/kg) completely prevented hypercholesterolemia. Decreases in plasma and hepatic cholesterol levels were strongly correlated with increased neutral sterol excretion. Ratios of neutral sterol excreted to pamaqueside administered were greater than 1:1 at all doses, in opposition to the formation of a stoichiometric complex previously suggested for tiqueside and other saponins. Ratios in tiqueside-treated rabbits were less than unity, a reflection of its lower potency. Pamaqueside-treated rabbits exhibited a more rapid decline in plasma cholesterol concentrations than control animals fed a cholesterol-free diet, indicating that the compound also inhibited the absorption of biliary cholesterol. Intravenous administration of pamaqueside had no effect on plasma cholesterol levels despite plasma levels twice those observed in rabbits given pamaqueside orally.These data indicate that pamaqueside and tiqueside induce hypocholesterolemia by blocking lumenal cholesterol absorption via a mechanism that apparently differs from the stoichiometric complexation of cholesterol hypothesized for other saponins.     

Sterol balance in hyperlipidemic patients after dietary exchange of carbohydrate for fat.

Dextrose was exchanged isocalorically for polyunsaturated fat in the liquid formula diets of 10 hyperlipidemic patients maintained under metabolic steady state conditions. Carbohydrate caused an increase of plasma triglycerides in all 10; plasma cholesterol rose in 7, and 6 of these 7 failed to show any increase in total fecal excretion of cholesterol. In contrast, fecal steroid excretion increased significantly in the three patients who maintained an unchanged or lower plasma cholesterol on the high-carbohydrate diet. Squalene, an obligatory precursor in the biosynthesis of cholesterol, rose in the plasma during carbohydrate feeding in 6 out of 6 patients studied. After a single intravenous infusion of radioactive cholesterol, plasma and feces were analyzed for specific activity over at least a four-month period. Plasma and fecal neutral sterol specific activity were essentially equivalent at all times in all patients, regardless of feeding regimen. On institution of carbohydrate feeding, the slope of cholesterol specific activity flattened for those seven patients who had a rise in plasma cholesterol concentration. There was no change in slope for the two patients with fixed plasma cholesterol levels nor for the one patient with a decreased plasma cholesterol on the high-carbohydrate diet. These experiments demonstrate a divergent response between plasma cholesterol concentration and cholesterol excretion. To establish causal relationship between the two (i.e., plasma cholesterol increases because excretion does not increase) will require further research. The flattening of specific activity die-away curves with rising cholesterol concentrations is best explained by mobilization of slowly turning-over tissue cholesterol into plasma. A subsequent decrease in cholesterol synthesis would also add to the slower decline of plasma cholesterol specific activity.     

Cholecystectomy prevents expansion of the bile acid pool and inhibition of cholesterol 7alpha-hydroxylase in rabbits fed cholesterol

To study the effect of cholecystectomy on the regulation of classic and alternative bile acid syntheses, gallbladder-intact (n = 20) and cholecystectomized (n = 20) New Zealand White rabbits were fed either chow or chow with 2% cholesterol (3 g/day). After 10 days, bile fistulas were constructed in half of each rabbit group to recover and measure the bile acid pool and biliary bile acid flux. After cholesterol feeding, the bile acid pool size increased from 268 +/- 55 to 444 +/- 77 mg (P < 0.01) with a 2-fold rise in the biliary bile acid flux in intact rabbits but did not expand the bile acid pool (270 +/- 77 vs. 276 +/- 62 mg), nor did the biliary bile acid flux increase in cholecystectomized rabbits. Ileal apical sodium-dependent bile acid transporter protein increased 46% from 93 +/- 6 to 136 +/- 23 units/mg (P < 0.01) in the intact rabbits but did not change in cholecystectomized rabbits (104 +/- 14 vs. 99 +/- 19 units/mg) after cholesterol feeding. Cholesterol 7alpha-hydroxylase activity was inhibited 59% (P < 0.001) while cholesterol 27-hydroxylase activity rose 83% (P < 0.05) after cholesterol feeding in the intact rabbits but neither enzyme activity changed significantly in cholesterol-fed cholecystectomized rabbits. Fecal bile acid outputs reflecting bile acid synthesis increased significantly in the intact but not in the cholecystectomized rabbits fed cholesterol.Removal of the gallbladder prevented expansion of the bile acid pool after cholesterol feeding as seen in intact rabbits because ileal bile acid transport did not increase. As a result, cholesterol 7alpha-hydroxylase was not inhibited.