The ABCG8 G574R variant, serum plant sterol levels, and cardiovascular disease risk in the Old Order Amish.

OBJECTIVE: To determine whether long-term exposure to moderate elevations in plasma plant sterol levels increases risk for atherosclerosis. METHODS AND RESULTS: In Old Order Amish participants aged 18 to 85 years, with (n=110) and without (n=181) 1 copy of the ABCG8 G574R variant, we compared mean plasma levels of plant sterols and cholesterol precursors and carotid intima-media wall thickness. Carriers of a single 574R allele had increased plant sterol levels (eg, 35%-37% higher plasma levels of sitosterol, campesterol, and stigmasterol) and increased plant sterol/cholesterol ratios (P<0.001 for all). 574R carriers had significantly decreased levels of lathosterol and lanosterol, precursors in a pathway for endogenous cholesterol synthesis, suggesting that plant sterols may alter regulation of genes involved in cholesterol synthesis. The G574R variant was not associated with high-density lipoprotein cholesterol or low-density lipoprotein cholesterol levels. Compared with noncarriers, 574R carriers had decreased carotid intima-media wall thickness (0.62 versus 0.66 mm; age- and sex-adjusted P=0.03). Adjustment for body weight, blood pressure, and standard lipid measures (high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides) did not alter this association. CONCLUSIONS: Although the G574R variant is associated with moderately elevated plant sterol levels, carriers of the 574R allele had modestly lower levels of carotid wall thickness compared with noncarriers.

Changes in cholesterol absorption and cholesterol synthesis caused by ezetimibe and/or simvastatin in men.

This study evaluates changes in cholesterol balance in hypercholesterolemic subjects following treatment with an inhibitor of cholesterol absorption or cholesterol synthesis or coadministration of both agents. This was a randomized, double blind, placebo-controlled, four-period crossover study to evaluate the effects of coadministering 10 mg ezetimibe with 20 mg simvastatin (ezetimibe/simvastatin) on cholesterol absorption and synthesis relative to either drug alone or placebo in 41 subjects. Each treatment period lasted 7 weeks. Ezetimibe and ezetimibe/simvastatin decreased fractional cholesterol absorption by 65% and 59%, respectively (P < 0.001 for both relative to placebo). Simvastatin did not significantly affect cholesterol absorption. Ezetimibe and ezetimibe/simvastatin increased fecal sterol excretion (corrected for dietary cholesterol), which also represents net steady state cholesterol synthesis, by 109% and 79%, respectively (P < 0.001). Ezetimibe, simvastatin, and ezetimibe/simvastatin decreased plasma LDL-cholesterol by 20, 38, and 55%, respectively. The coadministered therapy was well tolerated. The decreases in net cholesterol synthesis and increased fecal sterol excretion yielded nearly additive reductions in LDL-cholesterol for the coadministration of ezetimibe and simvastatin.

Cholesterol Absorption and Synthesis in Vegetarians and Omnivores.

SCOPE: Vegetarian diets are considered health-promoting; however, a plasma cholesterol lowering effect is not always observed. We investigate the link between vegetarian-diet-induced alterations in cholesterol metabolism. METHODS AND RESULTS: We study male and female omnivores, lacto-ovo vegetarians, lacto vegetarians, and vegans. Cholesterol intake, absorption, and fecal sterol excretion are measured as well as plasma concentrations of cholesterol and noncholesterol sterols. These serve as markers for cholesterol absorption, synthesis, and catabolism. The biliary cholesterol secretion rate is estimated. Flux data are related to body weight. Individual vegetarian diet groups are statistically compared to the omnivore group. Lacto vegetarians absorb 44% less dietary cholesterol, synthesized 22% more cholesterol, and show no differences in plasma total and LDL cholesterol. Vegan subjects absorb 90% less dietary cholesterol, synthesized 35% more cholesterol, and have a similar plasma total cholesterol, but a 13% lower plasma LDL cholesterol. No diet-related differences in biliary cholesterol secretion and absorption are observed. Total cholesterol absorption is lower only in vegans. Total cholesterol input is similar under all vegetarian diets. CONCLUSIONS: Unaltered biliary cholesterol secretion and higher cholesterol synthesis blunt the lowered dietary cholesterol intake in vegetarians. LDL cholesterol is significantly lower only in vegans.

Dietary plant sterols accumulate in the brain.

Dietary plant sterols and cholesterol have a comparable chemical structure. It is generally assumed that cholesterol and plant sterols do not cross the blood-brain barrier, but quantitative data are lacking. Here, we report that mice deficient for ATP-binding cassette transporter G5 (Abcg5) or Abcg8, with strongly elevated serum plant sterol levels, display dramatically increased (7- to 16-fold) plant sterol levels in the brain. Apolipoprotein E (ApoE)-deficient mice also displayed elevated serum plant sterol levels, which was however not associated with significant changes in brain plant sterol levels. Abcg5- and Abcg8-deficient mice were found to carry circulating plant sterols predominantly in high-density lipoprotein (HDL)-particles, whereas ApoE-deficient mice accommodated most of their serum plant sterols in very low-density lipoprotein (VLDL)-particles. This suggests an important role for HDL and/or ApoE in the transfer of plant sterols into the brain. Moreover, sitosterol upregulated apoE mRNA and protein levels in astrocytoma, but not in neuroblastoma cells, to a higher extend than cholesterol. In conclusion, dietary plant sterols pass the blood-brain barrier and accumulate in the brain, where they may exert brain cell type-specific effects.

The value of surrogate markers to monitor cholesterol absorption, synthesis and bioconversion to bile acids under lipid lowering therapies.

INTRODUCTION: Regulation of cholesterol (Chol) homeostasis is controlled by three main fluxes, i.e. intestinal absorption, de novo synthesis (ChS) and catabolism, predominantly as bile acid synthesis (BAS). High serum total Chol and LDL-Chol concentrations in particular are considered risk factors and markers for the development of atherosclerosis. Pharmaceutical treatments to lower serum Chol have focused on reducing absorption or ChS and increasing BAS. Monitoring of these three parameters is complex involving isotope techniques, cholesterol balance experiments and advanced mass spectrometry based analysis methods. Surrogate markers were explored that require only one single fasting blood sample collection. These markers were validated in specific, mostly physiological conditions and during statin treatment to inhibit ChS. They were also applied under cholesterol absorption restriction, but were not validated in this condition. We retrospectively evaluated the use of serum campesterol (Camp), sitosterol (Sit) and cholestanol (Cholol) as markers for cholesterol absorption, lathosterol (Lath) as marker for ChS and 7α-hydroxycholesterol (7α-OH-Ch) and 27-hydroxycholesterol (27-OH-Ch) as markers for BAS under conditions of Chol absorption restriction. Additionally, their values were corrected for Chol concentration (R_sterol or oxysterols). METHODS: Thirty-seven healthy male omnivore subjects were studied under treatments with placebo (PLAC), ezetimibe (EZE) to inhibit cholesterol absorption, simvastatin (SIMVA) to reduce cholesterol synthesis and a combination of both (EZE+SIMVA). Results were compared to those obtained in 18 pure vegetarian subjects (vegans) whose dietary Chol intake is extremely low. Relative or fractional Chol absorption (FrChA) was measured with the continuous feeding stable isotope procedure, ChS and BAS with the cholesterol balance method. The daily Chol intake (DICh) was inventoried and the daily Chol absorption (DACh) calculated. RESULTS: Monitoring cholesterol absorption, R_Camp, R_Sit and R_Cholol responded sensitively to changes in FrChA. R_Camp correlated well with FrChA in all omnivore treatment groups and in the vegan group. R_Camp confirmed reduced FrChA under EZE treatment and reduced DACh in the vegan subjects. R_Sit and R_Cholol did not accurately reflect FrChA or DACh in all situations. Monitoring endogenous cholesterol synthesis, R_Lath correlated with ChS in the vegan group, but in none of the omnivore treatment groups. R_Lath confirmed increased ChS under EZE treatment and was reduced under SIMVA treatment, while ChS was not. An increased ChS under EZE+SIMVA treatment could not be confirmed with R_Lath. R_Lath responded very insensitively to a change in ChS. Monitoring BAS, R_7α-OH-Ch but not R_27-OH-Ch correlated with BAS during PLAC, EZE and SIMVA treatments. In line with BAS, R_7α-OH-Ch did not differ in any of the omnivore treatment groups. R_7α-OH-Ch responded insensitively to a change in BAS. CONCLUSIONS: Under Chol absorption restriction, serum R_Camp is a sensitive and valid marker to monitor FrChA in a population with a normal DICh. Also, major changes in DACh can be detected in vegans. Serum R_Lath does not reflect ChS measured with the cholesterol balance method during EZE treatment. This result initiates the question whether the measured ChS reflects pure de novo synthesis. Serum R_7α-OH-Ch appears to be a valid but insensitive marker for BAS.

Impact of the SLCO1B1 polymorphism on the pharmacokinetics and lipid-lowering efficacy of multiple-dose pravastatin.

BACKGROUND AND OBJECTIVE: The polymorphism of SLCO1B1 (solute carrier organic anion transporter family, member 1B1), encoding the hepatic uptake transporter organic anion transporting polypeptide 1B1, has been associated with increased pravastatin concentrations in single-dose studies. We have investigated whether this polymorphism influences the pharmacokinetics and lipid-lowering efficacy of multiple-dose pravastatin. METHODS: A prospective, parallel-group study of 16 healthy volunteers, including 8 carriers of an SLCO1B1 variant haplotype and 8 control subjects, was carried out. Pravastatin pharmacokinetics and reduction in total and low-density lipoprotein (LDL) cholesterol concentrations were analyzed after treatment with 40 mg pravastatin daily for 3 weeks. RESULTS: The mean area under the plasma concentration-time curve of pravastatin was 110% higher (305.0 +/- 149.4 ng . h/mL [mean +/- SD] versus 144.9 +/- 48.2 ng . h/mL, P = .012) and the mean peak concentration in plasma was 231% higher (174.5 +/- 98.6 ng/mL versus 52.7 +/- 22.1 ng/mL, P = .0042) in the SLCO1B1 variant haplotype group than in the control group. Pravastatin significantly reduced the concentrations of total and LDL cholesterol in both groups. The mean percentage reduction in total cholesterol concentration was 13.1% +/- 9.1% and 19.1% +/- 8.3% in the variant and control groups, respectively (P = .19; 95% confidence interval of difference between groups, -15.3% to 3.3%). The mean percentage reduction in LDL cholesterol concentration was 27.7% +/- 8.3% in the variant group and 32.3% +/- 11.2% in the control group (P = .37; 95% confidence interval for difference, -15.1% to 6.0%). CONCLUSIONS: Despite considerably higher plasma pravastatin concentrations in carriers of an SLCO1B1 variant haplotype, there was no significant difference in the lipid-lowering efficacy of pravastatin between the variant haplotype and control groups. However, this pilot study had sufficient statistical power to detect only a large difference in lipid response between the 2 groups. Further clinical studies are warranted to characterize the impact of the SLCO1B1 polymorphism on the lipid response to pravastatin.

Intestinal expression of P-glycoprotein (ABCB1), multidrug resistance associated protein 2 (ABCC2), and uridine diphosphate-glucuronosyltransferase 1A1 predicts the disposition and modulates the effects of the cholesterol absorption inhibitor ezetimibe in humans.

BACKGROUND AND AIMS: Ezetimibe is an inhibitor of the cholesterol uptake transporter Niemann-Pick C1-like protein (NPC1L1). Target concentrations can be influenced by intestinal uridine diphosphate-glucuronosyltransferases (UGTs) and the efflux transporters P-glycoprotein (P-gp) (ABCB1) and multidrug resistance associated protein 2 (MRP2) (ABCC2). This study evaluates the contribution of these factors to the disposition and cholesterol-lowering effect of ezetimibe before and after induction of UGT1A1, P-gp, and MRP2 with rifampin (INN, rifampicin). METHODS: Serum concentrations of ezetimibe, as well as its glucuronide, and the plant sterols campesterol and sitosterol (surrogate for cholesterol absorption) were studied in 12 healthy subjects before and after rifampin comedication. In parallel, duodenal expression of UGT1A1, P-gp, MRP2, and NPC1L1 was quantified by use of real-time reverse transcriptase-polymerase chain reaction and quantitative immunohistochemical evaluation. The affinity of ezetimibe and its glucuronide to P-gp and MRP2 was assessed in P-gp- overexpressing Madin-Darby canine kidney II cells and P-gp-containing or MRP2-containing inside-out vesicles. RESULTS: Up-regulation of intestinal P-gp, MRP2, and UGT1A1 (but not of NPC1L1) by rifampin was associated with markedly decreased areas under the curve of ezetimibe and its glucuronide (116 +/- 78.1 ng.h/mL versus 49.9 +/- 31.0 ng.h/mL and 635 +/- 302 ng.h/mL versus 225 +/- 86.4 ng.h/mL, respectively; both P = .002) and increased intestinal clearances (2400 +/- 1560 mL/min versus 5500 +/- 4610 mL/min [P = .003] and 76.6 +/- 113 mL/min versus 316 +/- 457 mL/min [P = .010], respectively) and nearly abolished sterol-lowering effects. Intestinal expression of UGT1A1, ABCB1, and ABCC2 was inversely correlated with the effects of ezetimibe on plant sterol serum concentrations. Parallel in vitro studies confirmed that ezetimibe glucuronide is a high-affinity substrate of MRP2 and has a low affinity to P-gp whereas ezetimibe interacts with P-gp and MRP2. CONCLUSIONS: The disposition and sterol-lowering effects of ezetimibe are modified by metabolic degradation of the drug via intestinal UGT1A1 and either intestinal or hepatic secretion (or both) via P-gp and MRP2.

Genetic variant of the SREBF-1 gene is significantly related to cholesterol synthesis in man.

Sterol regulatory element binding proteins-1 and -2 (SREBPs) are transcription factors controlling lipid homeostasis in human cells. The G-allele carriers of the SREBF-1 gene C-G polymorphism in exon 18c and coding for glycine at the protein level (G952G) have shown to associate more frequently with obesity and type 2 diabetes than the C-allele carriers. However, the C-allele has suggested to be linked to dyslipidemia. Thus, our aim was to study effect of the SREBF-1 gene polymorphism (G952G) on sterol metabolism in man. Ninety-five subjects with moderate hypercholesterolemia participated in this study and 14 homozygous CC carriers of the SREBF-1 (G952G) gene were found. Plasma lathosterol concentration and lathosterol-to-cholesterol ratio, markers of endogenous cholesterol synthesis, were significantly higher in CC homozygous subject compared to others. Similarly muscle cholesterol (p=0.045) and lathosterol (p=0.054) concentrations were elevated in the CC homozygotes supporting the view that endogenous cholesterol synthesis rate is SREBF-1 genotype-dependent.

Sterol transporters: targets of natural sterols and new lipid lowering drugs.

Recent insights in the role of ATP-binding cassette (ABC) transporters ABCG5 and ABCG8, the discovery of ezetimibe, the first approved direct cholesterol absorption inhibitor, as well as the identification of Niemann-Pick C1 Like 1 (NPC1L1) protein as sterol transporter in the gut, focused attention on sterol transport processes in the small intestine and the liver. The identification of defective structures in the ABCG5 or ABCG8 transporters in patients with the rare disease of sitosterolemia elucidated their role as sterol efflux pumps regulating at least in parts the intestinal sterol absorption and the hepatic sterol output. ABCG5 and ABCG8 themselves are regulated by cholesterol via liver X receptors (LXRs), which are also activated by oxysterols and some derivatives of plant sterols. NPC1L1 could recently be identified as a major sterol transporter for the intestinal uptake of cholesterol as well as plant sterols. Studies in NPC1L1 knockout mice indicate that this transporter is essential for the intestinal uptake of sterols and that NPC1L1 might also be involved in the mechanism of action of ezetimibe. However, studies with photoreactive cholesterol as well as with photoreactive ezetimibe analogues suggest that other processes might also be involved in the mechanism of action of ezetimibe.

Influence of the SLCO1B1*1b and *5 haplotypes on pravastatin's cholesterol lowering capabilities and basal sterol serum levels.

We previously showed that variant SLCO1B1 haplotype *1b (A388G) accelerates and that *5 (T521C) delays hepatocellular uptake of the HMG-CoA reductase inhibitor pravastatin [Mwinyi et al. (2004): Clin Pharmacol Ther 75:415-421]. In the present study we checked for differential effects of variant SLCO1B1 haplotypes on hepatocellular cholesterol synthesis. We analyzed the serum levels of cholesterol, lathosterol, and campesterol in healthy white males which had been grouped on the basis of their SLCO1B1 haplotype: *1a (n=10), *1b (n=10), and *5 (n=8). The subjects received a single oral dose of 40 mg pravastatin. Cholesterol and lathosterol levels were lower in all subjects following pravastatin intake for up to 24. Median levels 6 h post-dosing of lathosterol decreased in each SLCO1B1 haplotype group in the rank order of *1b (-0.11 mg dl(-1); min-max: -0.20 to -0.04; p=0.005) > *1a (-0.09 mg dl(-1); min-max: -0.22 to -0.05; p=0.005) > *5 (-0.07 mg dl(-1); min-max: -0.17 to -0.05; p=0.012). Lathosterol median-change values were significantly greater in haplotype *1b than in haplotype *5 individuals (p=0.041, non-adjusted), which was congruent with the extent of mean changes in lathosterol-to-cholesterol ratios, although the latter did not reach statistical significance. Post-treatment serum levels of campesterol were not affected by SLCO1B1 haplotype. Interestingly, sterol basal serum levels tended to be highest in *1b carriers, followed by those in *1a and *5 individuals, with significant differences in lathosterol concentrations between the *1b and *5 (p=0.041, non-adjusted) haplotype group. Our findings suggest an association of SLCO1B1*1b and *5 haplotypes to pravastatin's inhibition of the hepatocellular HMG-CoA reductase. Furthermore, SLCO1B1 haplotypes seem to play a role in basal cholesterol homeostasis.

Polymorphism in ABCA1 influences CSF 24S-hydroxycholesterol levels but is not a major risk factor of Alzheimer's disease.

The ATP-binding cassette transporter A1 (ABCA1) mediates reverse cholesterol transport, polymorphisms have been shown to influence the levels of cholesterol and of HDL and the risk of coronary artery disease. Since altered cholesterol metabolism is also involved in Alzheimer's disease (AD), the effects of two ABCA1 polymorphisms (G-395C promoter polymorphism (rs 2246293) and exonic R219K) on the risk of AD in 241 AD patients and 294 non-demented controls, and on CSF cholesterol and 24S-hydroxycholesterol in 74 AD patients and 42 non-demented controls were investigated. None of the investigated ABCA1 polymorphisms influenced the risk of AD. However, the ABCA1 G-395C polymorphism influenced CSF levels of 24S-hydroxycholesterol, but not of cholesterol, whereas the R219K influenced neither CSF levels of 24S-hydroxycholesterol nor cholesterol. Our data support the observation that ABCA1 polymorphisms influence cholesterol metabolism of the brain, but might not act as a major risk factor in AD.

Inhibition of intestinal cholesterol absorption by ezetimibe in humans.

BACKGROUND: Ezetimibe has been shown to inhibit cholesterol absorption in animal models, but studies on cholesterol absorption in humans have not been performed thus far. METHODS AND RESULTS: The effect of ezetimibe (10 mg/d) on cholesterol absorption and synthesis, sterol excretion, and plasma concentrations of cholesterol and noncholesterol sterols was investigated in a randomized, double-blind, placebo-controlled, crossover study in 18 patients with mild to moderate hypercholesterolemia. Treatment periods lasted 2 weeks with an intervening 2-week washout period. Fractional cholesterol absorption rates averaged 49.8+/-13.8% on placebo and 22.7+/-25.8% on ezetimibe, indicating a reduction of 54% (geometric mean ratio; P< 0.001). Cholesterol synthesis increased by 89% from 931+/-1027 mg/d on placebo to 1763+/-1098 mg/d on ezetimibe (P<0.001), while the ratio of lathosterol-to-cholesterol, an indirect marker of cholesterol synthesis, was increased by 72% (P<0.001). Bile acid synthesis was insignificantly increased (placebo: 264+/-209 mg/d, ezetimibe: 308+/-184 mg/d; P=0.068). Mean percent changes from baseline for LDL and total cholesterol after ezetimibe treatment were -20.4% and -15.1%, respectively (P<0.001 for both), whereas campesterol and sitosterol were decreased by -48% and - 41%, respectively. CONCLUSION: In humans, ezetimibe inhibits cholesterol absorption and promotes a compensatory increase of cholesterol synthesis, followed by clinically relevant reductions in LDL and total cholesterol concentrations. Ezetimibe also reduces plasma concentrations of the noncholesterol sterols sitosterol and campesterol, suggesting an effect on the absorption of these compounds as well.

High-dose statins and skeletal muscle metabolism in humans: a randomized, controlled trial.

BACKGROUND: Myopathy, probably caused by 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition in skeletal muscle, rarely occurs in patients taking statins. This study was designed to assess the effect of high-dose statin treatment on cholesterol and ubiquinone metabolism and mitochondrial function in human skeletal muscle. METHODS: Forty-eight patients with hypercholesterolemia (33 men and 15 women) were randomly assigned to receive 80 mg/d of simvastatin (n = 16), 40 mg/d of atorvastatin (n = 16), or placebo (n = 16) for 8 weeks. Plasma samples and muscle biopsy specimens were obtained at baseline and at the end of the follow-up. RESULTS: The ratio of plasma lathosterol to cholesterol, a marker of endogenous cholesterol synthesis, decreased significantly by 66% in both statin groups. Muscle campesterol concentrations increased from 21.1 +/- 7.1 nmol/g to 41.2 +/- 27.0 nmol/g in the simvastatin group and from 22.6 +/- 8.6 nmol/g to 40.0 +/- 18.7 nmol/g in the atorvastatin group (P = .005, repeated-measurements ANOVA). The muscle ubiquinone concentration was reduced significantly from 39.7 +/- 13.6 nmol/g to 26.4 +/- 7.9 nmol/g (P = .031, repeated-measurements ANOVA) in the simvastatin group, but no reduction was observed in the atorvastatin or placebo group. Respiratory chain enzyme activities were assessed in 6 patients taking simvastatin with markedly reduced muscle ubiquinone and in matched subjects selected from the atorvastatin (n = 6) and placebo (n = 6) groups. Respiratory chain enzyme and citrate synthase activities were reduced in the patients taking simvastatin. CONCLUSIONS: High-dose statin treatment leads to changes in the skeletal muscle sterol metabolism. Furthermore, aggressive statin treatment may affect mitochondrial volume.

Cholesterol and plant sterol absorption: recent insights.

The recent discovery of transporters in the intestinal mucosa and the canalicular membrane has given new insights into the regulation of intestinal absorption as well as the biliary output of cholesterol and plant sterols. The 2 adenosine triphosphate (ATP)-binding cassette (ABC) half-transporters ABCG5 and ABCG8 are expressed in the mucosa cells and the canalicular membrane, and they resecrete sterols, especially absorbed plant sterols, back into the intestinal lumen and from the liver into bile. Defects of either of these cotransporters lead to the rare inherited disease of phytosterolemia, which is clinically defined by hyperabsorption and diminished biliary excretion of plant sterols. Furthermore, it has been recently demonstrated that the Niemann-Pick C1-Like 1 (NPC1L1) transporter is most likely responsible for the transport of cholesterol and plant sterols from the brush border membrane into the intestinal mucosa. Ezetimibe interferes with NPC1L1, reducing the intestinal uptake of cholesterol and plant sterols. These new findings contribute to our understanding of cholesterol and plant sterol concentrations in serum, and the effect of dietary and drug intervention to reduce serum concentrations of sterols.

Isotopomer spectral analysis of intermediates of cholesterol synthesis in patients with cerebrotendinous xanthomatosis.

Four patients with cerebrotendinous xanthomatosis (CTX) and 2 healthy controls received a constant proximal intraduodenal infusion of 1- 13 C-acetate as a stable-isotope-labeled marker of sterol synthesis. One patient was treated with pravastatin (20 mg twice daily) and another patient with chenodeoxycholic acid (250 mg tid). Every hour, venous blood and duodenal samples were obtained. Stable-isotope enrichment of neutral and polar sterols in serum and bile was assessed by gas chromatography/mass spectrometry. Isotopomer spectral analysis was performed on cholesterol, lathosterol, Delta-8-cholesterol, methylsterol, and lanosterol. Stable-isotope labeling of cholestanol, bile acids, and bile alcohols was analyzed by assessing the change over time of the ratio of M + 3 to M + 0. Eleven hours after marker infusion, we found up to 50% newly synthesized lathosterol in serum and up to 80% in bile, with similar results for other cholesterol precursors. In cholesterol, stable-isotope labeling could be demonstrated in all study subjects with a more prominent labeling in bile than in serum. No stable-isotope labeling was detected in cholestanol. Only minor stable-isotope incorporation was detectable in polar sterols in some subjects. Therapy with pravastatin did not have any effect on fractional or absolute synthesis rates or on the concentrations of cholestanol or cholesterol precursors compared to untreated patients with CTX. In contrast, therapy with chenodeoxycholic acid markedly lowered the concentrations of cholestanol and cholesterol precursors, led to a disappearance of bile alcohols, and reduced absolute synthesis rates of lathosterol. Isotopomer spectral analysis proved to be a powerful method to assess the endogenous synthesis of cholesterol precursors in patients with CTX. Higher fractional synthesis in bile than in serum may be due to the size of the pools in bile vs serum. Cholestanol exhibits no marker uptake and is therefore probably synthesized from preformed cholesterol. Biliary cholesterol secretion in patients with CTX is decreased compared to healthy controls.

Effect of raloxifene and hormone therapy on serum markers of brain and whole-body cholesterol metabolism in postmenopausal women.

OBJECTIVE: To compare the 2-year effects of raloxifene (Rlx) with oral postmenopausal hormone therapy (HT) on serum markers of brain and whole-body cholesterol metabolism. METHODS: In a randomized, double-blind, placebo-controlled trial, 95 healthy, non-hysterectomized, early postmenopausal women received either daily Rlx 60 mg (n = 24), Rlx 150 mg (n = 23), HT (conjugated equine estrogens 0.625 mg/medroxyprogesterone acetate 2.5 mg; n = 24), or placebo (n = 24). Fasting blood samples were collected at baseline and after 6, 12, and 24 months of treatment for measurement of serum concentrations of cholesterol by means of gas-liquid chromatography; 24S-hydroxycholesterol (cerebrosterol), lathosterol, and the plant sterol campesterol by means of gas-liquid chromatography-mass spectrometry. The analyses were performed retrospectively from serum samples stored at -70 degrees C for 5 years. RESULTS: Twenty-four months of treatment with raloxifene 150 mg was associated with a significant reduction in serum cholesterol concentrations (-10%, P = 0.007). The ratio of 24S-hydroxycholesterol to cholesterol, a serum marker of brain cholesterol metabolism, showed a significant increase after 6 and 12 months with raloxifene 150 mg but not after 24 months (P = 0.001). The ratio of lathosterol to cholesterol, a marker of whole-body cholesterol synthesis, increased with raloxifene 60 mg (P = 0.163), raloxifene 150 mg (P < 0.001), as well as with HT (P = 0.005). The ratio of campesterol to cholesterol, a marker of cholesterol absorption rate, was significantly reduced with HT (P = 0.002). CONCLUSION: Two-year treatment with raloxifene or HT had no influence on brain cholesterol metabolism, while whole-body cholesterol synthesis, assessed by the ratio of lathosterol to cholesterol, increased during raloxifene and HT.

Mutations in the human ATP-binding cassette transporters ABCG5 and ABCG8 in sitosterolemia.

Phytosterolemia or Sitosterolemia is a rare autosomal recessive disorder characterized by highly elevated plasma levels of plant sterols and cholesterol as a consequence of hyperabsorption and impaired biliary secretion of sterols. The disease is caused by mutations in two half size ATP-binding cassette transporters, ABCG5 and ABCG8. We have analyzed the genomic sequence of ABCG5 and ABCG8 in five well-characterized patients with Sitosterolemia. In the first patient we found a heterozygous mutation in exon 8 of the ABCG5 gene leading to a premature termination of the protein (Arg408Ter). This German patient is the first European showing a mutation of the ABCG5 gene. In a second patient we found a novel heterozygous mutation in exon 5 of ABCG8 (c.584T>A; Leu195Gln). Both patients were heterozygous for the identified mutation, but no mutation could be identified on the other chromosome. In three further analyzed patients we found mutations in exons 7, 9 and 11 of the ABCG8 gene, respectively, of which two result in a premature termination signal for translation products. One of these patients was compound heterozygous (Trp361Ter and Arg412Ter), the other was homozygous for Trp361Ter. The third patient was homozygous for an amino acid exchange (Gly574Arg). In conclusion this report describes one novel mutation affecting a highly conserved amino acid and two previously identified mutations in the ABCG8 gene. In addition, we identified for the first time a mutation in the ABCG5 gene of a European Sitosterolemia patient.

Reduction of plasma 24S-hydroxycholesterol (cerebrosterol) levels using high-dosage simvastatin in patients with hypercholesterolemia: evidence that simvastatin affects cholesterol metabolism in the human brain.

BACKGROUND: Previous studies have shown that patients with early onset of Alzheimer disease and vascular dementia have higher levels of circulating brain-derived 24S-hydroxycholesterol (cerebrosterol). Two recent epidemiological studies indicated that treatment with inhibitors of cholesterol synthesis (statins) reduces the incidence of Alzheimer disease. OBJECTIVE: To test the hypothesis that treatment with high-dosage simvastatin reduces circulating levels of 24S-hydroxycholesterol. DESIGN: Prospective, 24-week treatment trial for lowering of cholesterol levels. We conducted assessments at baseline, week 6, and week 24. SETTING: An academic outpatient clinical study. PATIENTS: Eighteen patients who met the criteria for hypercholesterolemia. INTERVENTION: Treatment with 80 mg/d of simvastatin at night. MAIN OUTCOME MEASURES: Plasma lipoprotein levels were measured enzymatically; lathosterol, by means of gas chromatography; and 24S-hydroxycholesterol, by means of gas chromatography-mass spectrometry. RESULTS: Simvastatin reduced total plasma cholesterol levels by 36% and 35% after 6 and 24 weeks, respectively (P<.001). Lathosterol levels were reduced by 74% and 72%, respectively, and the ratio of lathosterol to cholesterol, an indicator of whole-body cholesterol synthesis, was reduced by 60% and 61%, respectively (P<.001). Plasma 24S-hydroxycholesterol levels were lowered by 45% and 53%, respectively (P<.001). The ratio of 24S-hydroxycholesterol to cholesterol also decreased significantly (-12% [P=.01] and -23% [P<.002], respectively). The further reduction of 24S-hydroxycholesterol levels and its ratio to cholesterol from weeks 6 to 24 was also significant (P=.02 for both). CONCLUSIONS: The greater reduction of plasma concentrations of 24S-hydroxycholesterol compared with cholesterol indicates that simvastatin in a dosage of 80 mg/d reduces cholesterol turnover in the brain. The present results might describe a possible mechanism of how long-term treatment with statins could reduce the incidence of Alzheimer disease.

Reduction in levels of 24S-hydroxycholesterol by statin treatment in patients with Alzheimer disease.

BACKGROUND: The statin treatment of dyslipidemia is associated with a reduced risk of development of Alzheimer disease (AD). The effect may be mediated by a reduction in cholesterol biosynthesis in the brain, by lowering levels of apolipoprotein E (apo E)-containing lipoproteins, or by pleitropic effects such as reduction in beta-amyloid production. In the brain, cholesterol from damaged or dying neurons is converted to 24S-hydroxycholesterol by cholesterol 24-hydroxylase (CYP46). The oxysterol is subsequently transferred across the blood-brain barrier, transported to the liver by low-density lipoproteins (LDLs), and excreted as bile acids. Most of plasma 24S-hydroxycholesterol is derived from brain cholesterol; consequently, plasma levels of the oxysterol reflect brain cholesterol catabolism. OBJECTIVE: To examine the effect of 3 statins and a nonstatin hypolipidemic agent on plasma levels of 24S-hydroxycholesterol and apo E in patients with AD. STUDY DESIGN: The study had a sequential parallel design. It was open-labeled and involved lipoprotein and 24S-hydroxycholesterol evaluations at baseline and at 6 weeks of treatment with 40 mg of lovastatin, simvastatin, or pravastatin sodium per day, or 1 g of extended-release niacin per day. Blood samples were drawn after a 12-hour fast for measurement of plasma sterols, oxysterols, lipoprotein cholesterol, and levels of apo E, plasma transaminases, and glucose. Measurements were made at baseline and during treatment. RESULTS: Statin treatment reduced levels of plasma lathosterol by 49.5%, 24S-hydroxycholesterol by 21.4%, LDL cholesterol by 34.9%, and total cholesterol by 25%. The ratios of lathosterol-campesterol and 24S-hydroxycholesterol-LDL cholesterol were reduced significantly, but the ratio of 24S-hydroxycholesterol-total cholesterol was unchanged. Extended-release niacin also significantly reduced levels of 24S-hydroxycholesterol by 10% and LDL cholesterol by 18.1%. None of the agents lowered plasma concentration of apo E. CONCLUSIONS: Statins lowered levels of plasma 24S-hydroxycholesterol without affecting levels of apo E. The LDL lowering was more pronounced than 24S-hydroxycholesterol reductions. The effect of statins on LDL partially explains the reduction of plasma oxysterol level.

Cholesterol absorption inhibitors for the treatment of hypercholesterolaemia.

The benefits of lipid lowering therapy on coronary heart disease have been clearly established in many clinical trials on primary and secondary prevention. Despite the availability of potent lipid lowering drugs, many patients do not reach the current treatment goals. This paper reviews new therapeutic approaches in lipid lowering drugs focusing on compounds which lower cholesterol absorption. The role of plant sterols and stanols, new acyl-CoA:cholesterol O-acyl transferase (ACAT) inhibitors, microsomal triglyceride transfer protein (MTP) inhibitors, and ezetimibe are summarised. Although the lipid lowering effect of plant sterols and plant stanols is only moderate, their use as functional foods is beneficial for patients with mild hypercholesterolaemia and is able to enhance the lipid lowering effect of HMG-CoA reductase inhibitors (statins). The role of ACAT inhibitors that might also inhibit cholesterol absorption remains unclear. Avasimibe, the first oral bioavailable ACAT inhibitor, has entered phase III trials. However, the presently available data in humans do not indicate a clear clinical benefit. The role of MTP inhibitors, which exhibit remarkable effects on all plasma lipids, also remains unclear, as safety concerns must first be addressed. Ezetimibe, the first available 2-azetidinone, succeeded in phase III trials showing remarkable effects in inhibition of cholesterol absorption as well as cholesterol lowering. The synergistic effect of co-administration of ezetimibe with statins seemingly offers a new approach in reaching the therapeutic goals.