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Background and Aims Plant sterols are widely distributed in human diet but are poorly absorbed so that their plasma levels are very low. However, when fed in large amounts, they lower plasma cholesterol levels by interfering with cholesterol absorption. We have studied the effect of 4 weeks of feeding a chow diet supplemented with 1% plant sterols [brassicasterol (6.3%), campesterol (28.5%), stigmasterol (15.6%) and sitosterol (49.6%)], with or without SCH 58235 (a derivative of ezetimibe), 30 mg/kg per day, known to suppress intestinal cholesterol absorption, on plasma, tissue, biliary, and fecal sterols in Wistar and wild-type Kyoto (WKY) rats, and their metabolism by intestinal bacteria. Methods After 2 weeks of feeding control or experimental diet, rats were given [3α-3H]sitosterol intravenously and [4-14C]sitosterol by mouth, and blood was collected after 1, 2, 3, and 5 days after labeling to determine sitosterol absorption. Feces were collected during the last 3 days and freeze dried. At the end of feeding, bile fistulas were created in 3 rats of each strain and bile was collected for 1 hour. All rats were then sacrificed and plasma and liver were collected for sterol measurements and activities of hepatic HMG-CoA reductase, cholesterol 7α-hydroxylase, and cholesterol 27-hydroxylase. Results Wild-type Kyoto rats were hypercholesterolemic compared to Wistar rats and had increased plant sterols in the plasma. Plasma cholesterol tended to be lower in WKY rats after feeding with plant sterol-enriched diet whereas plant sterol levels rose to approximately 31% of plasma sterols in WKY and 14% in Wistar rats. However, brassicasterol and stigmasterol, with a double bond at C-22, constituted less than 3.5% of total plasma plant sterols. After feeding, biliary plant sterols increased 2.25-fold in Wistar and 1.5-fold in WKY rats, suggesting less hepatic clearance in WKY rats. SCH 58235 feeding significantly increased plasma as well as biliary cholesterol levels in both the untreated and plant sterol-fed WKY rats, and the plasma plant sterols showed a tendency to increase but did not reach significant level. Intestinal bacteria in both rat strains metabolized all plant sterols to mainly the 5β-H-stanols. However, the C-22 double bond was stable to bacterial degradation. Intestinal absorption of sitosterol and cholesterol was increased 1.5- and 1.3-fold, respectively, in the WKY rats as compared to the Wistar rats, and plant sterol feeding lowered absorption of these sterols in both strains. Absorption of both these sterols was also lowered in SCH 58235-treated rats in both strains and was further lowered when SCH 58235 and plant sterols were simultaneously fed. The activity of the rate-limiting enzyme, HMG-CoA reductase, was increased 1.57-fold in Wistar rats and 1.27-fold in WKY rats that were fed plant sterols as compared to untreated rats. Conclusions (1) Plant sterol absorption was increased whereas hepatic elimination of all sterols was diminished in WKY rats accounting for elevated cholesterol and plant sterol levels. (2) The 1% plant sterol-enriched diet tended to lower plasma cholesterol levels whereas SCH 58235 feeding significantly increased plasma cholesterol levels in the WKY rats. (3) Intestinal absorption of sterols with C-22 double bond is diminished and the side-chain double bond is resistant to intestinal bacteria.

We investigated the effect of SC-435, a competitive inhibitor of ileal apical sodium-dependent bile acid cotransporter (ASBT) on ileal bile acid absorption and the hepatic nuclear receptor FXR (farnesoid X receptor), which regulates cholesterol 7 alpha-hydroxylase (CYP7A1) activity and mRNA levels. Eighteen New Zealand White (NZW) rabbits were divided into 2 groups: controls (n = 10) and fed SC-435 125 mg/kg/d for 1 week (n = 8). In rabbits treated with SC-435, fecal bile acid outputs increased by more than 8 times, reflecting substantial bile acid malabsorption. Plasma cholesterol levels decreased 26%, while bile acid pool sizes and biliary bile acid outputs did not change after treatment. CYP7A1 activity increased 64% and mRNA rose by 4 times after treatment. The expression of FXR target genes in the liver, short heterodimer partner (SHP) and bile salt export pump (BSEP), decreased 11.6 and 2.6 times, respectively, after treatment, which indicates inactivation of hepatic FXR. However, the mRNA levels of ileal bile acid binding protein (IBABP) did not change significantly, while ileal ASBT mRNA expression increased by 2.4 times after treatment. Rabbits treated with SC-435 developed ileal bile acid malabsorption, which decreased the return of bile acids (FXR ligands) to the liver to inactivate hepatic FXR, which upregulated CYP7A1 and lowered plasma cholesterol levels. Although fecal bile acid malabsorption was substantial, increased bile acid production from hepatic cholesterol kept biliary bile acid outputs intact. Thus, a new balance was reached in the liver, where increased bile acid synthesis compensated for diminished ileal bile acid absorption to maintain the circulating enterohepatic bile acid pool.

We compared the effect of treatments with hydrophilic bile acids (ursodeoxycholic and ursocholic acids), cholestyramine, and lovastatin versus chenodeoxycholic acid in 4 patients with cerebrotendinous xanthomatosis (CTX). Bile acids and bile alcohols in plasma, bile, and urine before and after treatment were quantitated by gas-liquid chromatography. Untreated, all patients showed abnormal biliary bile acid composition: cholic acid (72.7%) and chenodeoxycholic acid (6.2%), and polyhydroxylated C 27 -bile alcohols (10.0%), and elevated plasma cholestanol levels. Treatment with hydrophobic chenodeoxycholic acid inhibited abnormal bile acid synthesis (virtual disappearance of C 27 -bile alcohols from plasma, bile, and urine and marked reduction of plasma cholestanol levels). Hydrophilic ursodeoxycholic and ursocholic acids did not inhibit abnormal bile acid synthesis, while cholestyramine increased abnormal bile acid synthesis (continued increased formation of polyhydroxylated C 27 -bile alcohols and further elevation of plasma cholestanol levels). Lovastatin did not affect abnormal bile acid synthesis or reduce plasma cholestanol levels. The results demonstrate that impaired side-chain oxidation in bile acid synthesis due to mutations of Cyp27 results in increased formation of polyhydroxylated C 27 -bile alcohols and cholestanol in CTX. Hydrophobic chenodeoxycholic acid, but not cholestyramine, lovastatin, or hydrophilic 7β-hydroxy acids, inhibited the abnormal synthetic pathway. The role of chenodeoxycholic acid in downregulating abnormal bile acid synthesis in CTX is emphasized.

Sitosterolemia is a rare, recessively inherited disease characterized clinically by accelerated atherosclerosis and xanthomas and biochemically by hyperabsorption and retention of sitosterol and other plant sterols in tissues. Decreased cholesterol biosynthesis and inhibition of 3-hydroxy-3-methylgluratyl coenzyme A (HMG-CoA) reductase and other enzymes in the biosynthetic pathway have been associated with enhanced low-density lipoprotein (LDL) receptor function. We examined the effects of cholesterol and sitosterol on sterol concentrations and composition and HMG-CoA reductase activity in monocyte-derived macrophages (MDM) from 12 control and 3 homozygous sitosterolemic subjects. The cells were cultured up to 7 days in media devoid of plant sterols, but containing increasing amounts of serum cholesterol. Before culture, MDM from the homozygous sitosterolemic subjects contained 22% more total sterols than cells from control subjects. Plant sterols and stanols represented 15.6% of MDM total sterols in sitosterolemic cells, but only 3.8% in control cells. After 7 days of culture in 10% delipidated serum (DLS) (20 [mu ]g/mL cholesterol, no sitosterol), all plant sterols were eliminated so that cells from both phenotypes contained only cholesterol. When DLS was replaced with fetal bovine serum (FBS) (300 [mu ]g/mL cholesterol), with and without addition of 200 [mu ]g/mL LDL, cholesterol levels in MDM from sitosterolemic subjects increased 108% (P [lt ] .05) compared with a 65% increase (P [lt ] .04) in control MDM cultured similarly. MDM HMG-CoA reductase activity from the 3 sitosterolemic subjects, which was significantly lower than controls at baseline (24 [plusmn] 3 v 60 [plusmn] 10 pmol/mg/min, P [lt ] .05), was not downregulated by increased cellular cholesterol levels, as observed in control cells. Control MDM were also cultured in medium that contained 10% DLS and was supplemented with 100 [mu ]g/mL cholesterol or sitosterol dissolved in ethanol or the ethanol vehicle alone. In contrast to cellular cholesterol accumulation, which significantly downregulated HMG-CoA reductase activity ([minus ]53%, P [lt ] .05), the increase in cellular sitosterol up to 25.1% of total sterols did not change MDM HMG-CoA reductase activity. Evidence of a normal HMG-CoA reductase protein in sitosterolemic cells, which was not derepressed upon removal of cellular sitosterol, and the failure of cellular sitosterol to inhibit normal HMG-CoA reductase activity argue against feedback inhibition by sitosterol as a mechanism for low reductase activity in this disease. The larger accumulation of sterols and inadequate downregulation of HMG-CoA reductase in MDM may be mechanisms for foam cell formation and explain, in part, the increased risk of atherosclerosis in sitosterolemia.

We compared hepatic cholesterol metabolism in apolipoprotein (apo) E-knockout (KO) mice with their wild-type counterparts. We also investigated the effects of treatment with phytosterols or probucol on the activity of hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase (cholesterol synthesis), cholesterol 7 alpha-hydroxylase and sterol 27-hydroxylase (bile acid synthesis), and low-density lipoprotein (LDL) receptor function in this animal model of atherogenesis. These findings were then related to treatment-induced changes in plasma, hepatic, and fecal sterol concentrations. Mouse liver membranes have binding sites similar to LDL receptors; the receptor-mediated binding represents 80% of total binding and is LDL concentration-dependent. These binding sites have higher affinity for apo E-containing particles than apo B only-containing particles. Deletion of apo E gene was associated with several-fold increases in plasma cholesterol levels, 1.5-fold increase in hepatic cholesterol concentrations, 50% decrease in HMG-CoA reductase activity, 30% increase in cholesterol 7 alpha-hydroxylase and 25% decrease in LDL receptor function. Treatment of apo E-KO mice with either probucol or phytosterols significantly reduced plasma cholesterol levels. Phytosterols significantly increased the activity of hepatic HMG-CoA reductase, and probucol significantly increased cholesterol 7 alpha-hydroxylase activity. Neither treatment significantly altered hepatic LDL receptor function. Phytosterols, but not probucol, significantly increased fecal sterol excretion and decreased hepatic cholesterol concentrations. Plasma cholesterol lowering effects of phytosterols and probucol are due to different mechanisms: stimulation of cholesterol catabolism via increased bile acid synthesis by probucol and decreased cholesterol absorption by phytosterols. In the absence of apo E, hepatic LDL receptors could not be upregulated and did not contribute to the cholesterol lowering effects of either agent.

The regulation of the classic and alternative bile acid synthetic pathways by key hepatic enzyme activities (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase, respectively) was examined in bile acid depletion and replacement and cholesterol-feeding experiments with rats, guinea pigs, and rabbits. The bile acid pool was depleted by creating a bile fistula (BF) and collecting bile for 2 to 5 days, and it was replaced by intraduodenal infusion of the major biliary bile acids (taurocholic acid [TCA], glycochenodeoxycholic acid [GCDCA], and glycocholic acid [GCA] in the rat, guinea pig, and rabbit, respectively) at rates equivalent to the measured hepatic flux of the bile acids. To study the effects of cholesterol, the animals were fed for 7 days on a basal diet with and without 2% cholesterol. Cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities, measured by isotope incorporation assays, were related to bile acid output and composition and hepatic cholesterol concentrations. Intraduodenal infusion of bile acids increased the output of the tested bile acids, but did not significantly change hepatic cholesterol concentrations and had no effect on sterol 27-hydroxylase activity. Neither bile acid depletion nor replacement affected sterol 27-hydroxylase activity when three different substrates (cholesterol, 5beta-cholestane-3alpha,7alpha-diol, and 5beta-cholestane-3alpha,7alpha,12alpha-triol) were tested. In contrast, feeding 2% cholesterol increased hepatic cholesterol concentrations in rats, guinea pigs, and rabbits threefold, twofold, and eightfold, respectively, and increased hepatic mitochondrial sterol 27-hydroxylase activity (conversion of cholesterol to 27-hydroxycholesterol) in all three animal models. The stimulation and feedback inhibition of cholesterol 7alpha-hydroxylase activity by bile acid depletion and replacement were observed in all three animal models, whereas the effect of cholesterol feeding was species-dependent (cholesterol 7alpha-hydroxylase activity increased in the rat, did not change in the guinea pig, and was inhibited in the rabbit). Thus, in contrast to sterol 27-hydroxylase, which was upregulated by cholesterol but not affected by bile acid depletion and replacement in all three animal models, cholesterol 7alpha-hydroxylase activity was controlled consistently and inversely by the hepatic flux of bile acids, but was species-dependent in its response to a 1-week feeding with 2% cholesterol.

Campestanol (24-methyl-5alpha-cholestan-3beta-ol) is a naturally occurring plant stanol, structurally similar to cholesterol (5-cholesten-3beta-ol) and widely distributed in vegetable oils consumed in human diets. We measured the absorption and turnover of campestanol by the plasma dual-isotope ratio method and mathematical analysis of specific activity versus time decay curves after simultaneous oral and intravenous pulse-labeling with [3alpha-3H]- and [23-14C]-labeled campestanol, respectively, in New Zealand White (NZW) rabbits: six fed chow and six fed chow with 125 mg/d campestanol and 175 mg/d sitostanol (24-ethyl-5alpha-cholestan-3beta-ol). Plasma concentrations increased insignificantly from 0.08+/-0.01 to 0.09+/-0.01 mg/dL with dietary stanols. The percent campestanol absorption measured by the plasma dual-isotope ratio method after the rabbits were fasted for 6 hours yielded the percent absorption in the absence of competing intestinal sterols and stanols and declined insignificantly from 11.6%+/-3.5% in controls to 8.1%+/-3.7% in the treated rabbit groups. In contrast, the turnover, which measured actual absorption averaged over 24 hours, increased from 0.12+/-0.05 to 0.37+/-0.05 mg/d (P < .05) with campestanol and sitostanol added to the diet. However, the actual percent absorption declined from 3% to 0.3% of dietary intake with the campestanol and sitostanol-enriched diet. Campestanol pool sizes, although remaining small, increased slightly from 1.1+/-0.4 to 2.5+/-1.5 mg. The removal constant (KA) from pool A (MA) did not change significantly with added dietary campestanol and sitostanol (KA= -0.040+/-0.005 v -0.037+/-0.007 d(-1)). The results demonstrate small campestanol plasma concentrations and body pools even when the rabbits consumed substantial amounts because (1) intestinal absorption was limited and (2) was further reduced by competing dietary sitostanol, and (3) campestanol was removed rapidly from the body. Thus, campestanol, which shares the same basic structure and intestinal absorption pathway with cholesterol, does not accumulate when fed, and may be incorporated into the diet to block cholesterol absorption.

We examined the relationship between brain sterol composition and associative learning (classical conditioning of the eyeblink response) in newly weaned rats fed BM 15.766 (BM) for 4 months. This compound inhibits 7-dehydrocholesterol-delta7-reductase, which catalyzes the conversion of 7-dehydrocholesterol to cholesterol, the last step in the synthetic pathway. As countertreatment, half of the BM-treated rats were fed 2% cholesterol during the last 2 months. With BM, cholesterol concentrations declined 91% in plasma, but with cholesterol feeding, the levels increased 50% compared with baseline values. 7-Dehydrocholesterol, which was not detected at baseline, increased to 55% of plasma sterols with BM but decreased to 5% of total plasma sterols when cholesterol was added. With BM, brain cholesterol levels decreased 60% and did not increase after cholesterol was added. However, 7-dehydrocholesterol, which comprised 39% of brain sterols with BM, decreased to 31% (P.05) when cholesterol was fed. Hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase activity in the liver increased 2.2-fold with BM and declined 95% after adding cholesterol, but did not change in the brain. BM treatment for 4 months prevented learning of the conditioned eyeblink response as compared with controls. In contrast, BM-treated rats supplemented with cholesterol acquired the conditioned eyeblink response. Chronic inhibition of 7-dehydrocholesterol-delta7-reductase reduced cholesterol and increased 7-dehydrocholesterol levels in plasma and brain, and was associated with impaired learning. Cholesterol feeding corrected plasma and hepatic sterol levels and reduced brain 7-dehydrocholesterol concentrations to reestablish normal learning.

We investigated the effect of the hepatic hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin and the primary bile acid chenodeoxycholic acid (CDCA) on plasma sterol and bile alcohol concentrations and the excretion of bile alcohols in urine in a 38-year-old homozygote with cerebrotendinous xanthomatosis (CTX). Untreated, plasma cholesterol concentrations were less than normal (171 +/- 5 v 185 +/- 3 mg/dL, P < .05) while plasma cholestanol levels were more than 20 times higher than the control mean (2.26 +/- 0.17 v 0.1 +/- 0.1 mg/dL, P < .0001). Plasma and urinary bile alcohol concentrations were markedly increased (12.6 +/- 0.6 and 154 micrograms/mL, respectively, v trace amounts in controls), with the ratio of 5 beta-cholestane-3 alpha,7 alpha,12 alpha, 25-tetrol to 5 beta-cholestane, 3 alpha,7 alpha,12 alpha,23 (22 and 24),25-pentols being 1.6 in plasma and reversed to 0.15 in urine. Treatment with lovastatin (40 mg/d) reduced plasma cholesterol concentrations 13%, but failed to decrease plasma cholestanol or bile alcohol levels. Abundant amounts of bile alcohols continued to be excreted in urine. In contrast, CDCA (750 mg/d) inhibited abnormal bile acid synthesis, as evidence by a 17-fold decrease in total bile alcohol levels in plasma and a 29-fold decrease in urine and the virtual elimination of cholic acid and deoxycholic acid from the bile. Plasma cholestanol concentrations also decreased 85%, but cholesterol levels increased 14%. The combination of CDCA with lovastatin did not improve plasma cholestanol or bile alcohol concentrations compared with CDCA treatment alone.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the mechanism for reduced cholesterol biosynthesis in sitosterolemia with xanthomatosis. The conversion of acetate to cholesterol and total and active hydroxymethylglutaryl (HMG) coenzyme A (CoA) reductase activities, enzyme protein mass, and catalytic efficiency were related to low-density lipoprotein (LDL) receptor function in freshly isolated mononuclear leukocytes collected at 9 AM after a 12-hour fast from two affected sisters and 12 control subjects. Active HMG-CoA reductase activity was determined in mononuclear leukocyte microsomes prepared and assayed in the presence of sodium fluoride, while total HMG-CoA reductase activity was determined in the absence of the phosphatase inhibitor. Enzyme protein was assayed using rabbit polyclonal anti-rat liver microsomal HMG-CoA reductase serum. The rates at which [14C]acetate was transformed to cholesterol by sitosterolemic mononuclear leukocytes were decreased 29% and 41%, respectively, compared with the mean value for mononuclear leukocytes from 12 control subjects. Similarly, total HMG-CoA reductase activities were 71% and 68% lower in sitosterolemic mononuclear leukocyte microsomes and were associated with 62% and 65% less enzyme protein than the mean for the control microsomal preparations. This marked decrease in HMG-CoA reductase protein mass in sitosterolemic microsomes was partially compensated for by an increase in the proportion of active enzyme. Sitosterolemic plasma and mononuclear leukocyte cholesterol concentrations were not significantly different from control values, although total sterol levels were increased about 20% because of abundant plant sterols. In contrast, receptor-mediated LDL degradation by sitosterolemic mononuclear leukocytes was increased 50% over control.(ABSTRACT TRUNCATED AT 250 WORDS)

Sitosterol and cholesterol metabolism were studied in a patient with coexisting phytosterolemia and cholestanolemia, and in a control subject, both on similar diets containing about 170 mg cholesterol and 135 mg phytosterols per day. The turnover of 22,23-3H-sitosterol and 4-14C-cholesterol, given intravenously, were followed for up to 372 days. The specific activity-time curves for both sterols were resolved into two exponentials and fitted into a two-pool model. The half-lives of both exponential curves for sitosterol, in the patient, were abnormally long. Equilibration of the tracer between the two pools, in the patient, occurred at about 30 days as compared to 10-15 days in the control subject. The daily turnover of sitosterol in the patient was estimated to be 10 times greater than that in the control subject. The patient's total body exchangeable pool of sitosterol was 9.6 g or about 80 times the amount found in the control. The patient's plasma phytosterol levels fell by 25% when he went on a diet containing only 10 mg phytosterols per day. During this period the specific activity of his plasma sitosterol with respect to an equilibrated dose of 3H-labeled tracer remained constant; this was compatible with the absence of endogenous synthesis. Cholesterol turnover in the patient showed prolonged half-lives for both exponential curves and reduced fractional daily loss from the fast-exchanging pool. The patient's xanthoma sterols underwent 16% and 55% exchange with plasma sitosterol and cholesterol, respectively, on day 60, indicating the presence of a third exchangeable pool.