Degree of serum LDL cholesterol reduction by simvastatin and ezetimibe is dependent on baseline LDL cholesterol concentration but not on baseline values and changes in cholesterol synthesis and absorption parameters.

OBJECTIVE: We questioned whether the baseline status of low-density lipoprotein cholesterol (LDL-C), cholesterol synthesis and absorption, and the changes in these parameters determine the change in serum LDL-C under statin or ezetimibe treatment or under combination treatment. MATERIALS AND METHODS: 37 mildly hypercholesterolemic healthy male subjects were studied under placebo, simvastatin (20 mg/d), ezetimibe (10 mg/d), and combination treatment. We correlated the change of LDL-C (ΔLDL-C) under treatment with the placebo end values of LDL-C (baseline), whole-body cholesterol synthesis, and hepatic cholesterol synthesis (serum lathosterol to cholesterol ratio) as well as fractional absorption rate (FAR) of cholesterol and serum campesterol to cholesterol ratio. The change in serum LDL-C was also correlated with the changes in synthesis and absorption parameters. RESULTS: ΔLDL-C was highly negatively related to baseline LDL-C under ezetimibe (p < 0.0001), simvastatin (p < 0.0001), and combination treatment (p < 0.0001). Under combination treatment, LDL-C lowering appears possible from baseline values of 10 mg/dL upwards, while ΔLDL-C was independent of the baseline value (-50 to -60%). ΔLDL-C was positively associated with placebo FAR under ezetimibe (p = 0.0106) and combination treatment (p = 0.0457). No associations were found between ΔLDL-C and baseline values for synthesis nor between ΔLDL-C and changes in synthesis and absorption surrogate markers. CONCLUSION: Under ezetimibe, simvastatin, and combination treatment, ΔLDL-C is predominantly dependent on the baseline LDL-C concentration. We hypothesize that the concentration gradient between serum LDL-C and hepatic cellular cholesterol determines the efficiency of serum LDL-C lowering. Combination treatment is the preferred treatment.

Lemon Flavonoid Extract Eriomin Improves Pro/Antioxidant Status and Interferes with Cholesterol Metabolism without Affecting Serum Cholesterol Levels in Aged Rats.

This study aimed to assess the antioxidant capacity of lemon flavonoid extract Eriomin® (LE) and its impact on cholesterol metabolism in the context of healthy aging. We orally treated 24-month-old male Wistar rats with an LE (40 mg/kg) suspended in 0.3 mL of sunflower oil. At the same time, control groups received an equal volume of sunflower oil (CON) or remained untreated (ICON) daily for 4 weeks. We examined LE's effects on superoxide dismutase and catalase- and glutathione-related enzyme activities, the concentration of lipid peroxides and protein carbonyls, total oxidant status (TOS) and antioxidant status (TAS), and oxidative stress index (OSI) in the liver, jejunum, and ileum. We also measured total cholesterol, its biosynthetic precursors (lanosterol, lathosterol, desmosterol), its degradation products (bile acid precursors) in the serum, liver, jejunum, and ileum, and serum phytosterols (intestinal absorption markers). LE reduced TOS, TAS, and OSI (p < 0.05) compared with control values, indicating its consistent antioxidant action in all examined organs. LE lowered hepatic desmosterol (p < 0.05) while also reducing 7α- and 24-hydroxycholesterol levels in the liver and ileum (p < 0.01). Serum cholesterol, hepatic gene expression, and the immunostaining intensity of CYP7A1 were unchanged. In conclusion, LE exerted non-enzymatic antioxidant effects and reduced cholesterol degradation, reducing its biosynthesis products, thereby maintaining serum cholesterol levels.

Dynamics of the enterohepatic circulation of bile acids in healthy humans.

Regulation of bile acid metabolism is normally discussed as the regulation of bile acid synthesis, which serves to compensate for intestinal loss in order to maintain a constant pool size. After a meal, bile acids start cycling in the enterohepatic circulation. Farnesoid X receptor-dependent ileal and hepatic processes lead to negative feedback inhibition of bile acid synthesis. When the intestinal bile acid flux decreases, the inhibition of synthesis is released. The degree of inhibition of synthesis and the mechanism and degree of activation are still unknown. Moreover, in humans, a biphasic diurnal expression pattern of bile acid synthesis has been documented, indicating maximal synthesis around 3 PM and 9 PM. Quantitative data on the hourly synthesis schedule as compensation for intestinal loss are lacking. In this review, we describe the classical view on bile acid metabolism and present alternative concepts that are based on the overlooked feature that bile acids transit through the enterohepatic circulation very rapidly. A daily profile of the cycling and total bile acid pool sizes and potential controlled and uncontrolled mechanisms for synthesis are predicted. It remains to be elucidated by which mechanism clock genes interact with the Farnesoid X receptor-controlled regulation of bile acid synthesis. This mechanism could become an attractive target to enhance bile acid synthesis at night, when cholesterol synthesis is high, thus lowering serum LDL-cholesterol.

Anti-PCSK 9 antibodies increase the ratios of the brain-specific oxysterol 24S-hydroxycholesterol to cholesterol and to 27-hydroxycholesterol in the serum.

AIMS: The serum ratios of the brain-specific oxysterol 24S-hydroxycholesterol (24S-OHC) to cholesterol and to 27-OHC reflect brain cholesterol turnover. We studied the effect of proprotein convertase subtilisin/kexin type 9 monoclonal antibodies (PCSK9ab) that enhance low-density lipoprotein receptor activity on serum cholesterol and oxysterol concentrations. METHODS: Twenty-eight hypercholesterolaemic patients (15 males and 13 females) responding insufficiently to maximally tolerated statin and/or ezetimibe therapy were additionally subcutanously treated biweekly with either the PCSK9ab alirocumab (150 mg, n = 13) or evolocumab (140 mg, n = 15). Fasting serum cholesterol was measured by gas chromatography and the oxysterols 24S-OHC and 27-OHC using gas chromatography-mass spectrometry before, after 1-month (n = 28) and after 3-month (n = 13) treatment. RESULTS: As expected, PCSK9ab treatment lowered serum cholesterol and oxysterol levels after 1 month. The serum ratio of 24S-OHC to cholesterol increased after 1 month by 17 ± 28% (mean ± standard deviation; 95% confidence interval [CI]: 5.8 to 28%; P < .01) and 24S-OHC to 27-OHC by 15 ± 39% (95% CI: 0.2 to 30%; P < .01). Within 3 months, 24S-OHC to cholesterol increased by 2.8 µg g-1  mo-1 (95% CI: 2.1 to 3.6; P < .01) and 24S-OHC to 27-OHC by 0.019 mo-1 (95% CI: 0.007 to 0.032; P < .01). CONCLUSION: The serum ratios of 24S-OHC to cholesterol and to 27-OHC increased after treatment with PCSK9ab. We hypothesize that this is caused by a reduced entrance of 27-OHC into the brain, increased synthesis of brain cholesterol, increased production of 24S-OHC and its secretion across the blood-brain barrier.

Serum 4ß-hydroxycholesterol increases during fluconazole treatment.

PURPOSE: The antifungal drugs ketoconazole and itraconazole reduce serum concentrations of 4ß-hydroxycholesterol, which is a validated marker for hepatic cytochrome P450 (CYP) 3A4 activity. We tested the effect of another antifungal triazole agent, fluconazole, on serum concentrations of different sterols and oxysterols within the cholesterol metabolism to see if this inhibitory reaction is a general side effect of azole antifungal agents. METHODS: In a prospective, double-blind, placebo-controlled, two-way crossover design, we studied 17 healthy subjects (nine men, eight women) who received 400 mg fluconazole or placebo daily for 8 days. On day 1 before treatment and on day 8 after the last dose, fasting blood samples were collected. Serum cholesterol precursors and oxysterols were measured by gas chromatography-mass spectrometry-selected ion monitoring and expressed as the ratio to cholesterol (R_sterol). RESULTS: Under fluconazole treatment, serum R_lanosterol and R_24,25-dihydrolanosterol increased significantly without affecting serum cholesterol or metabolic downstream markers of hepatic cholesterol synthesis. Serum R_4ß-, R_24S-, and R_27-hydroxycholesterol increased significantly. CONCLUSION: Fluconazole inhibits the 14α-demethylation of lanosterol and 24,25-dihydrolanosterol, regulated by CYP51A1, without reduction of total cholesterol synthesis. The increased serum level of R_4ß-hydroxycholesterol under fluconazole treatment is in contrast to the reductions observed under ketoconazole and itraconazole treatments. The question, whether this increase is caused by induction of CYP3A4 or by inhibition of the catabolism of 4ß-hydroxycholesterol, must be answered by mechanistic in vitro and in vivo studies comparing effects of various azole antifungal agents on hepatic CYP3A4 activity.

Laxative treatment with polyethylene glycol decreases microbial primary bile salt dehydroxylation and lipid metabolism in the intestine of rats.

Polyethylene glycol (PEG) is a frequently used osmotic laxative that accelerates gastrointestinal transit. It has remained unclear, however, whether PEG affects intestinal functions. We aimed to determine the effect of PEG treatment on intestinal sterol metabolism. Rats were treated with PEG in drinking water (7%) for 2 wk or left untreated (controls). We studied the enterohepatic circulation of the major bile salt (BS) cholate with a plasma stable isotope dilution technique and determined BS profiles and concentrations in bile, intestinal lumen contents, and feces. We determined the fecal excretion of cholesterol plus its intestinally formed metabolites. Finally, we determined the cytolytic activity of fecal water (a surrogate marker of colorectal cancer risk) and the amount and composition of fecal microbiota. Compared with control rats, PEG treatment increased the pool size (+51%; P < 0.01) and decreased the fractional turnover of cholate (-32%; P < 0.01). PEG did not affect the cholate synthesis rate, corresponding with an unaffected fecal primary BS excretion. PEG reduced fecal excretion of secondary BS and of cholesterol metabolites (each P < 0.01). PEG decreased the cytolytic activity of fecal water [54 (46-62) vs. 87 (85-92)% erythrocyte potassium release in PEG-treated and control rats, respectively; P < 0.01]. PEG treatment increased the contribution of Verrucomicrobia (P < 0.01) and decreased that of Firmicutes (P < 0.01) in fecal flora. We concluded that PEG treatment changes the intestinal bacterial composition, decreases the bacterial dehydroxylation of primary BS and the metabolism of cholesterol, and increases the pool size of the primary BS cholate in rats.

From Dietary Cholesterol to Blood Cholesterol.

The Nutrients' Special Issue "From dietary cholesterol to blood cholesterol" aims to supply existing knowledge and novel new research data about human cholesterol (C) fluxes [...].

Measurement of Serum Low Density Lipoprotein Cholesterol and Triglyceride-Rich Remnant Cholesterol as Independent Predictors of Atherosclerotic Cardiovascular Disease: Possibilities and Limitations.

The serum low density lipoprotein cholesterol (LDL-C) concentration is the dominant clinical parameter to judge a patient's risk of developing cardiovascular disease (CVD). Recent evidence supports the theory that cholesterol in serum triglyceride-rich lipoproteins (TRLs) contributes significantly to the atherogenic risk, independent of LDL-C. Therefore, combined analysis of both targets and adequate treatment may improve prevention of CVD. The validity of TRL-C calculation is solely dependent on the accuracy of the LDL-C measurement. Direct measurement of serum LDL- C is more accurate than established estimation procedures based upon Friedewald, Martin-Hopkins, or Sampson equations. TRL-C can be easily calculated as total C minus high density lipoprotein C (HDL-C) minus LDL-C. Enhanced serum LDL-C or TRL-C concentrations require different therapeutic approaches to lower the atherogenic lipoprotein C. This review describes the different atherogenic lipoproteins and their possible analytical properties and limitations.

A non-invasive, low-cost study design to determine the release profile of colon drug delivery systems: a feasibility study.

PURPOSE: Conventional bioavailability testing of dosage forms based on plasma concentration-time graphs of two products in a two-period, crossover-design, is not applicable to topical treatment of intestinal segments. We introduce an isotope dual-label approach ((13)C- and (15)N(2)-urea) for colon drug delivery systems that can be performed in a one-day, non-invasive study-design. METHODS: Four healthy volunteers took an uncoated or a ColoPulse-capsule containing (13)C-urea and an uncoated capsule containing (15)N(2)-urea. In case of colon-release (13)C-urea is fermented and (13)C detected as breath (13)CO(2). Absorbed (13)C-urea and (15)N-urea are detected in urine. RESULTS: C and (15)N in urine released from uncoated capsules showed a ratio of 1.01 ± 0.06. The (13)C/(15)N-recovery ratio after intake of a ColoPulse-capsule was constant and lower >12 h post-dose (median 0.22, range 0.13-0.48). The (13)C/(15)N-ratio in a single urine sample at t ≥ 12 h predicted the 24 h non-fermented fraction (13)C of <26 %. Breath (13)CO(2) indicated delayed (>3 h) release and a fermented fraction (13)C >54 %. CONCLUSIONS: Breath and urine (13)C and (15)N data describe the release-profile and local bioavailability of a colon delivery device. This allows non-invasive bioavailability studies for evaluation of colon-specific drug delivery systems without radioactive exposure and with increased power and strongly reduced costs.

Laxative treatment with polyethylene glycol does not affect lipid absorption in rats.

OBJECTIVES: Polyethylene glycol (PEG) is a frequently used laxative agent. It is unknown, however, whether PEG affects the absorptive capacity of the intestine. Reduced lipid (dietary fat and cholesterol) absorption induced by long-term PEG treatment could negatively affect growth in children. We tested whether PEG accelerates gastrointestinal transit and alters lipid absorption and plasma lipid levels. METHODS: Wistar rats were administered drinking water with or without PEG (7%) for 2 weeks. We studied whole gut transit time by recording the first appearance of red feces after intragastric carmine red administration. We measured plasma concentrations of cholesterol and triglycerides, dietary fat absorption by 48-hour fat balance and by plasma appearance of intragastrically administered stable-isotope labeled fats, and cholesterol absorption with a dual stable isotope technique. RESULTS: PEG decreased whole gut transit time by 20% (P=0.028) without causing diarrhea. PEG treatment did neither affects overall dietary fat balance nor fat uptake kinetics, cholesterol absorption, or plasma lipid concentrations. CONCLUSIONS: PEG does not affect lipid absorption nor steady-state plasma lipid levels in rats, although it accelerates the gastrointestinal transit.

Film coated tablets (ColoPulse technology) for targeted delivery in the lower intestinal tract: influence of the core composition on release characteristics.

The design of a film coating technology which allows a tablet to deliver the drug in the ileocolonic segment would offer new treatment possibilities. The objective is to develop a platform technology that is suitable for a broad range of drug compounds. We developed a coated tablet with a delayed, pulsatile release profile based on a pH-sensitive coating technology (ColoPulse). The production process was validated, and the effect of core composition on the in vitro release and water uptake investigated. The release profile of the standard tablet core composition, based on the use of cellulose as a filler, was independent of the coat thickness in a range of 9.0-13.2 mg/cm(2). The release profile of a coated tablet was strongly influenced when cellulose was partly replaced by the model substance glucose (loss of sigmoidal release), citric acid (stabilization), sodium bicarbonate (destabilization) or sodium benzoate (destabilization). The film coating takes up water when below the pH-threshold. However, this did not cause early disintegration of the coating. The ColoPulse technology is successfully applied on tablets. The in vitro release characteristics of the coated tablets are influenced by the composition of the core.

From Dietary Cholesterol to Blood Cholesterol, Physiological Lipid Fluxes, and Cholesterol Homeostasis.

Dietary cholesterol (C) is a major contributor to the endogenous C pool, and it affects the serum concentration of total C, particularly the low-density lipoprotein cholesterol (LDL-C). A high serum concentration of LDL-C is associated with an increased risk for atherosclerosis and cardiovascular diseases. This concentration is dependent on hepatic C metabolism creating a balance between C input (absorption and synthesis) and C elimination (conversion to bile acids and fecal excretion). The daily C absorption rate is determined by dietary C intake, biliary C secretion, direct trans-intestinal C excretion (TICE), and the fractional C absorption rate. Hepatic C metabolism coordinates C fluxes entering the liver via chylomicron remnants (CMR), LDL, high-density lipoproteins (HDL), hepatic C synthesis, and those leaving the liver via very low-density lipoproteins (VLDL), biliary secretion, and bile acid synthesis. The knowns and the unknowns of this C homeostasis are discussed.

Serum Low Density Lipoprotein Cholesterol Concentration Is Not Dependent on Cholesterol Synthesis and Absorption in Healthy Humans.

Introduction. Pharmacological reduction of cholesterol (C) synthesis and C absorption lowers serum low-density lipoprotein C (LDL-C) concentrations. We questioned whether high baseline C synthesis or C absorption translates into high serum LDL-C concentrations or if there was no connection. Therefore, we studied the association between serum LDL-C and C synthesis or C absorption in healthy subjects. Methods. Three published data sets of young subjects on different diets (study 1), mildly hypercholesterolemic subjects without cardiovascular disease (study 2) and healthy controls of the Framingham study (study 3) were used. The three study populations varied in sex, age, and weight. C synthesis and C fractional absorption rate (FAR) were measured with fecal sterol balance and stable isotope techniques (studies 1 and 2). Additionally, serum lathosterol and campesterol concentrations corrected for the serum total C concentration (R_lathosterol and R_campesterol) were used as markers for hepatic C synthesis and C FAR, respectively (studies 1−3). Linear regression analysis was applied to evaluate associations between LDL-C, C synthesis, and C absorption. Results. Seventy-three, 37, and 175 subjects were included in studies 1, 2, and 3, respectively. No statistically significant associations were found between LDL-C and the measured C synthesis and C FAR, nor for R_lathosterol and R_campesterol in any of the study groups. This lack of associations was confirmed by comparing the male subjects of studies 1 and 2. Study 1 subjects had a 50% lower serum LDL-C than the study 2 subjects (p < 0.01), but not a lower C synthesis, C FAR, R-lathosterol, or R_campesterol. Conclusions. Under physiological conditions, C synthesis and C FAR are not major determinants of circulating serum LDL-C concentrations in healthy subjects. The results need to be confirmed in large-scale studies in healthy subjects and patients at risk for cardiovascular disease.

Recent Advances in the Digestive, Metabolic and Therapeutic Effects of Farnesoid X Receptor and Fibroblast Growth Factor 19: From Cholesterol to Bile Acid Signaling.

Bile acids (BA) are amphiphilic molecules synthesized in the liver (primary BA) starting from cholesterol. In the small intestine, BA act as strong detergents for emulsification, solubilization and absorption of dietary fat, cholesterol, and lipid-soluble vitamins. Primary BA escaping the active ileal re-absorption undergo the microbiota-dependent biotransformation to secondary BA in the colon, and passive diffusion into the portal vein towards the liver. BA also act as signaling molecules able to play a systemic role in a variety of metabolic functions, mainly through the activation of nuclear and membrane-associated receptors in the intestine, gallbladder, and liver. BA homeostasis is tightly controlled by a complex interplay with the nuclear receptor farnesoid X receptor (FXR), the enterokine hormone fibroblast growth factor 15 (FGF15) or the human ortholog FGF19 (FGF19). Circulating FGF19 to the FGFR4/ß-Klotho receptor causes smooth muscle relaxation and refilling of the gallbladder. In the liver the binding activates the FXR-small heterodimer partner (SHP) pathway. This step suppresses the unnecessary BA synthesis and promotes the continuous enterohepatic circulation of BAs. Besides BA homeostasis, the BA-FXR-FGF19 axis governs several metabolic processes, hepatic protein, and glycogen synthesis, without inducing lipogenesis. These pathways can be disrupted in cholestasis, nonalcoholic fatty liver disease, and hepatocellular carcinoma. Thus, targeting FXR activity can represent a novel therapeutic approach for the prevention and the treatment of liver and metabolic diseases.

Improved glycemic control with colesevelam treatment in patients with type 2 diabetes is not directly associated with changes in bile acid metabolism.

UNLABELLED: Bile acids (BAs) are essential for fat absorption and appear to modulate glucose and energy metabolism. Colesevelam, a BA sequestrant, improves glycemic control in type 2 diabetes mellitus (T2DM). We aimed to characterize the alterations in BA metabolism associated with T2DM and colesevelam treatment and to establish whether metabolic consequences of T2DM and colesevelam are related to changes in BA metabolism. Male subjects with T2DM (n = 16) and controls (n = 12) were matched for age and body mass index. BA pool sizes and synthesis/input rates were determined before and after 2 and 8 weeks of colesevelam treatment. T2DM subjects had higher cholic acid (CA) synthesis rate, higher deoxycholic acid (DCA) input rate, and enlarged DCA pool size. Colesevelam resulted in a preferential increase in CA synthesis in both groups. CA pool size was increased whereas chenodeoxycholic acid and DCA pool sizes were decreased upon treatment. Fasting and postprandial fibroblast growth factor 19 (FGF19) levels did not differ between controls and diabetics, but were decreased by treatment in both groups. Colesevelam treatment reduced hemoglobin A1C by 0.7% (P < 0.01) in diabetics. Yet, no relationships between BA kinetic parameters and changes in glucose metabolism were found in T2DM or with colesevelam treatment. CONCLUSION: Our results reveal significant changes in BA metabolism in T2DM, particularly affecting CA and DCA. Colesevelam treatment reduced FGF19 signaling associated with increased BA synthesis, particularly of CA, and resulted in a more hydrophilic BA pool without altering total BA pool size. However, these changes could not be related to the improved glycemic control in T2DM.

Bile salt sequestration induces hepatic de novo lipogenesis through farnesoid X receptor- and liver X receptor alpha-controlled metabolic pathways in mice.

UNLABELLED: Diabetes is characterized by high blood glucose levels and dyslipidemia. Bile salt sequestration has been found to improve both plasma glycemic control and cholesterol profiles in diabetic patients. Yet bile salt sequestration is also known to affect triglyceride (TG) metabolism, possibly through signaling pathways involving farnesoid X receptor (FXR) and liver X receptor alpha (LXRalpha). We quantitatively assessed kinetic parameters of bile salt metabolism in lean C57Bl/6J and in obese, diabetic db/db mice upon bile salt sequestration using colesevelam HCl (2% wt/wt in diet) and related these to quantitative changes in hepatic lipid metabolism. As expected, bile salt sequestration reduced intestinal bile salt reabsorption. Importantly, bile salt pool size and biliary bile salt secretion remained unchanged upon sequestrant treatment due to compensation by de novo bile salt synthesis in both models. Nevertheless, lean and db/db mice showed increased, mainly periportally confined, hepatic TG contents, increased expression of lipogenic genes, and increased fractional contributions of newly synthesized fatty acids. Lipogenic gene expression was not induced in sequestrant-treated Fxr(-/-) and Lxralpha(-/-) mice compared with wild-type littermates, in line with reports indicating a regulatory role of FXR and LXRalpha in bile salt-mediated regulation of hepatic lipid metabolism. CONCLUSION: Bile salt sequestration by colesevelam induces the lipogenic pathway in an FXR- and LXRalpha-dependent manner without affecting the total pool size of bile salts in mice. We speculate that a shift from intestinal reabsorption to de novo synthesis as source of bile salts upon bile salt sequestration affects zonation of metabolic processes within the liver acinus.

Unconjugated bile salts shuttle through hepatocyte peroxisomes for taurine conjugation.

UNLABELLED: Bile acid-CoA:amino acid N-acyltransferase (BAAT) conjugates bile salts to glycine or taurine, which is the final step in bile salt biosynthesis. In addition, BAAT is required for reconjugation of bile salts in the enterohepatic circulation. Recently, we showed that BAAT is a peroxisomal protein, implying shuttling of bile salts through peroxisomes for reconjugation. However, the subcellular location of BAAT remains a topic of debate. The aim of this study was to obtain direct proof for reconjugation of bile salts in peroxisomes. Primary rat hepatocytes were incubated with deuterium-labeled cholic acid (D(4)CA). Over time, media and cells were collected and the levels of D(4)CA, D(4)-tauro-CA (D(4)TCA), and D(4)-glyco-CA (D(4)GCA) were quantified by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Subcellular accumulation of D(4)-labeled bile salts was analyzed by digitonin permeabilization assays and subcellular fractionation experiments. Within 24 hours, cultured rat hepatocytes efficiently (>90%) converted and secreted 100 µM D(4)CA to D(4)TCA and D(4)GCA. The relative amounts of D(4)TCA and D(4)GCA produced were dependent on the presence of glycine or taurine in the medium. Treatment of D(4)CA-exposed hepatocytes with 30-150 µg/mL digitonin led to the complete release of D(4)CA, D(4)GCA, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cytosolic marker). Full release of D(4)TCA, catalase, and BAAT was only observed at 500 µg/mL digitonin, indicating the presence of D(4)TCA in membrane-enclosed organelles. D(4)TCA was detected in fractions of purified peroxisomes, which did not contain D(4)CA and D(4)GCA. CONCLUSION: We established a novel assay to study conjugation and intra- and transcellular transport of bile salts. Using this assay, we show that cholic acid shuttles through peroxisomes for taurine-conjugation.

Applications of stable isotopes in clinical pharmacology.

This review aims to present an overview of the application of stable isotope technology in clinical pharmacology. Three main categories of stable isotope technology can be distinguished in clinical pharmacology. Firstly, it is applied in the assessment of drug pharmacology to determine the pharmacokinetic profile or mode of action of a drug substance. Secondly, stable isotopes may be used for the assessment of drug products or drug delivery systems by determination of parameters such as the bioavailability or the release profile. Thirdly, patients may be assessed in relation to patient-specific drug treatment; this concept is often called personalized medicine. In this article, the application of stable isotope technology in the aforementioned three areas is reviewed, with emphasis on developments over the past 25 years. The applications are illustrated with examples from clinical studies in humans.

ABCG5/G8 polymorphisms and markers of cholesterol metabolism: systematic review and meta-analysis.

Genetic variation at the ABCG5/G8 locus has been associated with markers of cholesterol homeostasis. As data originate from small-scale studies, we performed a meta-analysis to study these associations in a large dataset. We first investigated associations between five common ABCG5/G8 polymorphisms (p.Q604E, p.D19H, p.Y54C, p.T400K, and p.A632V) and plasma sterol levels in 245 hypercholesterolaemic individuals. No significant associations were found. Subsequently, our data were pooled into a meta-analysis that comprised 3,364 subjects from 16 studies (weighted mean age, 46.7 ± 10.5 years; BMI, 23.9 ± 3.5 kg/m(2)). Presence of the minor 632V allele correlated with reduced LDL-C concentrations (n = 367) compared with homozygosity for the 632A variant [n = 614; -0.11 mmol/l (95% CI, range: -0.20 to -0.02 mmol/l); P = 0.01]. The remaining polymorphisms were not associated with plasma lipid levels. Carriers of the 19H allele exhibited lower campesterol/TC (n = 83; P < 0.001), sitosterol/TC (P < 0.00001), and cholestanol/TC (P < 0.00001), and increased lathosterol/TC ratios (P = 0.001) compared with homozygous 19D allele carriers (n = 591). The ABCG8 632V variant was associated with a clinically irrelevant LDL-C reduction, whereas the 19H allele correlated with decreased cholesterol absorption and increased synthesis without affecting the lipid profile. Hence, associations between frequently studied missense ABCG5/G8 polymorphisms and markers of cholesterol homeostasis are modest at best.

Intestinal FXR-mediated FGF15 production contributes to diurnal control of hepatic bile acid synthesis in mice.

Hepatic bile acid synthesis is subject to complex modes of transcriptional control, in which the bile acid-activated nuclear receptor farnesoid X receptor (FXR) in liver and intestine-derived, FXR-controlled fibroblast growth factor 15 (Fgf15) are involved. The Fgf15 pathway is assumed to contribute significantly to control of hepatic bile acid synthesis. However, scientific evidence supporting this assumption is primarily based on gene expression data. Using intestine-selective FXR knockout mice (iFXR-KO), we show that contribution of intestinal FXR-Fgf15 signalling in regulation of hepatic cholesterol 7α-hydroxylase (Cyp7A1) expression depends on time of the day with increased hepatic Cyp7A1 expression in iFXR-KO mice compared with controls exclusively during the dark phase. To assess the physiological relevance hereof, we determined effects of intestine-selective deletion of FXR on physiological parameters such as bile formation and kinetics of the enterohepatic circulation of bile acids. It appeared that intestinal FXR deficiency leads to a modest but significant increase in cholic acid pool size, without changes in fractional turnover rate. As a consequence, bile flow and biliary bile acid secretion rates were increased in iFXR-KO mice compared with controls. Feeding a bile acid-containing diet or treatment with a bile acid sequestrant similarly affected bile formation in iFXR-KO and control mice and induced similar changes in Cyp7A1 and Cyp8B1 expression patterns. In conclusion, this study is the first to demonstrate the physiological relevance of the contribution of the intestinal FXR-Fgf15 signalling pathway in control of hepatic bile acid synthesis. Fgf15 contributes to the regulation of hepatic bile acid synthesis in mice mainly during the dark phase. Expansion of the circulating bile acid pool as well as bile acid sequestration diminishes the contribution of intestinal FXR-Fgf15 signalling in control of hepatic bile acid synthesis and bile formation.