Supplementation of a dairy drink enriched with milk phospholipids in patients with atopic dermatitis - a double-blind, placebo-controlled, randomized, cross-over study.

BACKGROUND & AIMS: Reduced epidermal ceramide content may lead to an impaired skin barrier in atopic dermatitis. Plasma concentration of the ceramide precursor sphingomyelin increases after milk-fat consumption due to affected lipoprotein metabolism, although sphingomyelin, a main component of milk phospholipids, might also directly influence plasma sphingomyelin levels. The aim was to determine whether supplementation of a dairy drink with milk phospholipids improves skin parameters and influences plasma lipid profile. METHODS: In a double-blind cross-over study, 39 patients were randomized into 2 groups and daily received phospholipid milk (3 g phospholipids ≙ 0.75 g sphingomyelin) or normal whole milk as placebo control for 6 weeks. SCORAD indices, serum immune and plasma lipid parameters were determined. RESULTS: SCORAD indices did not differ between groups following control and phospholipid milk supplementation (control milk: 10.9 ± 5.9 vs. phospholipid milk: 11.7 ± 6.9, P = 0.416), but were significantly decreased compared to baseline (baseline: 15.6 ± 8.8, P < 0.05). Plasma sphingomyelin proportions were also similar after the treatments (control milk: 27.5 ± 2.3 vs. phospholipid milk: 27.4 ± 2.6% of total phospholipids, P = 0.894), but were significantly increased compared to baseline (20.7 ± 2.4% of total phospholipids, P < 0.05). CONCLUSIONS: Supplementation of a dairy drink with milk phospholipids has no beneficial effect on skin parameters compared to consumption of whole milk in patients with atopic dermatitis. To elucidate an impact of the plasma sphingomyelin proportion on skin conditions, further studies are necessary. Clinical trial ID: Registered under ClinicalTrials.gov. Identifier no. NCT01326520.

Lupin protein isolate versus casein modifies cholesterol excretion and mRNA expression of intestinal sterol transporters in a pig model.

BACKGROUND: Lupin proteins exert hypocholesterolemic effects in man and animals, although the underlying mechanism remains uncertain. Herein we investigated whether lupin proteins compared to casein modulate sterol excretion and mRNA expression of intestinal sterol transporters by use of pigs as an animal model with similar lipid metabolism as humans, and cellular cholesterol-uptake by Caco-2 cells. METHODS: Two groups of pigs were fed cholesterol-containing diets with either 230 g/kg of lupin protein isolate from L. angustifolius or 230 g/kg casein, for 4 weeks. Faeces were collected quantitatively over a 5 d period for analysis of neutral sterols and bile acids by gas chromatographically methods. The mRNA abundances of intestinal lipid transporters were analysed by real-time RT-PCR. Cholesterol-uptake studies were performed with Caco-2 cells that were incubated with lupin conglutin γ, phytate, ezetimibe or albumin in the presence of labelled [4-14C]-cholesterol. RESULTS: Pigs fed the lupin protein isolate revealed lower cholesterol concentrations in total plasma, LDL and HDL than pigs fed casein (P < 0.05). Analysis of faeces revealed a higher output of cholesterol in pigs that were fed lupin protein isolate compared to pigs that received casein (+57.1%; P < 0.05). Relative mRNA concentrations of intestinal sterol transporters involved in cholesterol absorption (Niemann-Pick C1-like 1, scavenger receptor class B, type 1) were lower in pigs fed lupin protein isolate than in those who received casein (P < 0.05). In vitro data showed that phytate was capable of reducing the uptake of labelled [4-14C]-cholesterol into the Caco-2 cells to the same extend as ezetimibe when compared to control (-20.5% vs. -21.1%; P < 0.05). CONCLUSIONS: Data reveal that the cholesterol-lowering effect of lupin protein isolate is attributable to an increased faecal output of cholesterol and a reduced intestinal uptake of cholesterol. The findings indicate phytate as a possible biofunctional ingredient of lupin protein isolate.

Bile acids and gut peptide secretion after bariatric surgery: a 1-year prospective randomized pilot trial.

OBJECTIVE: Increased delivery of bile acid salts (BA) to distal L-cells and altered TGR5 receptor activation may contribute to the early and substantial increases in gut peptide secretion seen after bariatric surgery. To further elucidate a potential role of BA in the secretion of GLP-1 and PYY, we analyzed plasma BA concentrations in 14 morbidly obese patients undergoing gastric bypass or sleeve gastrectomy in a prospective, randomized 1-year trial. DESIGN AND METHODS: Patients received a standard test meal and blood was collected before and after eating, prior to, and 1 week, 3 months, and 12 months after surgery. RESULTS: Pre-surgery, basal BA concentrations were significantly lower in bariatric patients than in healthy controls. One year post-surgery, bariatric patients expressed variably increased BA concentrations (gastric bypass patients ∼2 fold increase, P ≤ 0.05). However, whereas in both patient groups, marked increases in GLP-1 and PYY and improved glycemic control was seen already 1 week and 3 months post-surgery, changes in plasma BA followed a different pattern: basal and postprandial plasma BA concentrations increased much slower, more progressively with significant increases only 1-year post-surgery. CONCLUSIONS: Based on these findings, BA do not appear to be key mediators of the early increase in GLP-1 and PYY response in post-bariatric patients.

Dietary crystalline common-, micro-, nanoscale and emulsified nanoscale sitosterol reduce equally the cholesterol pool in guinea pigs, but varying nanosystems result in different sterol concentrations in serosal jejunum.

Due to hypocholesterolemic effects, sitosterol is used in functional foods and nanoscale dispersions. To investigate the influence of dietary sitosterol on sterol concentrations in Dunkin Hartley guinea pigs, seven groups consisting of eight animals each were fed either a basal diet (BD), a high-cholesterol diet (HC) or a high-cholesterol diet supplemented with crystalline commonscale (CCS), microscale (CMS, low-dosed: CMLS), nanoscale (CNS) or emulsified nanoscale (ENS) sitosterol. When compared to HC group, all sitosterol formulations decreased liver cholesterol concentrations. No differences in cholesterol or sitosterol concentration were found in plasma and liver between CCS, CMS, CNS, and ENS groups. Apparent cholesterol digestibility decreased by increasing crystalline microscale sitosterol doses. Despite a lower sitosterol intake, ENS group had higher serosal sitosterol concentrations in jejunum than CNS group. To elucidate an impact of the sitosterol nanosystem on gut tissues further studies are necessary. FROM THE CLINICAL EDITOR: In this study, the use of sitosterols in a rat model led to contradicting conclusions regarding their ability to reduce cholesterol levels efficiently in guinea pigs, suggesting that more preclinical data is needed before this method could become applicable to human studies.

Milk phospholipid and plant sterol-dependent modulation of plasma lipids in healthy volunteers.

PURPOSE: Hypolipidemic and/or hypocholesterolemic effects are presumed for dietary milk phospholipid (PL) as well as plant sterol (PSt) supplementation. The aim was to induce changes in plasma lipid profile by giving different doses of milk PL and a combination of milk PL with PSt to healthy volunteers. METHODS: In an open-label intervention study, 14 women received dairy products enriched with moderate (3 g PL/day) or high (6 g PL/day) dose of milk PL or a high dose of milk PL combined with PSt (6 g PL/day + 2 g PSt/day) during 3 periods each lasting 10 days. RESULTS: Total cholesterol concentration and HDL cholesterol concentration were reduced following supplementation with 3 g PL/day. No significant change in LDL cholesterol concentration was found compared with baseline. High PL dose resulted in an increase of LDL cholesterol and unchanged HDL cholesterol compared with moderate PL dose. The LDL/HDL ratio and triglyceride concentration remained constant within the study. Except for increased phosphatidyl ethanolamine concentrations, plasma PL concentrations were not altered during exclusive PL supplementations. A combined high-dose PL and PSt supplementation led to decreased plasma LDL cholesterol concentration, decreased PL excretion, increased plasma sphingomyelin/phosphatidyl choline ratio, and significant changes in plasma fatty acid distribution compared with exclusive high-dose PL supplementation. CONCLUSION: Milk PL supplementations influence plasma cholesterol concentrations, but without changes of LDL/HDL ratio. A combined high-dose milk PL and PSt supplementation decreases plasma LDL cholesterol concentration, but it probably enforces absorption of fatty acids or fatty acid-containing hydrolysis products that originated during lipid digestion.

C57Bl/6 N mice on a western diet display reduced intestinal and hepatic cholesterol levels despite a plasma hypercholesterolemia.

BACKGROUND: Small intestine and liver greatly contribute to whole body lipid, cholesterol and phospholipid metabolism but to which extent cholesterol and phospholipid handling in these tissues is affected by high fat Western-style obesogenic diets remains to be determined. METHODS: We therefore measured cholesterol and phospholipid concentration in intestine and liver and quantified fecal neutral sterol and bile acid excretion in C57Bl/6 N mice fed for 12 weeks either a cholesterol-free high carbohydrate control diet or a high fat Western diet containing 0.03% (w/w) cholesterol. To identify the underlying mechanisms of dietary adaptations in intestine and liver, changes in gene expression were assessed by microarray and qPCR profiling, respectively. RESULTS: Mice on Western diet showed increased plasma cholesterol levels, associated with the higher dietary cholesterol supply, yet, significantly reduced cholesterol levels were found in intestine and liver. Transcript profiling revealed evidence that expression of numerous genes involved in cholesterol synthesis and uptake via LDL, but also in phospholipid metabolism, underwent compensatory regulations in both tissues. Alterations in glycerophospholipid metabolism were confirmed at the metabolite level by phospolipid profiling via mass spectrometry. CONCLUSIONS: Our findings suggest that intestine and liver react to a high dietary fat intake by an activation of de novo cholesterol synthesis and other cholesterol-saving mechanisms, as well as with major changes in phospholipid metabolism, to accommodate to the fat load.

Intestinal steroid profiles and microbiota composition in colitic mice.

Reduced gut microbiota diversity in conjunction with a bloom of few bacterial species is a common feature in inflammatory bowel disease (IBD) patients. However, the environmental changes caused by inflammation and their possible impact on the microbiota are largely unknown. Since IBD is associated with an impaired intestinal steroid metabolism, we hypothesized that changes in intestinal steroid and particularly bile acid (BA) concentrations affect microbial communities. We used Interleukin-10 deficient (IL-10-/-) mice as a model for chronic gut inflammation. Healthy wild-type mice served as controls. In these animals, intestinal steroid concentrations and gut microbial diversity were analyzed at 24 weeks of age. The IL 10-/- mice developed moderate inflammation in cecum and colon and colorectal tumor formation was observed in 55 % of the animals. Compared to the healthy conditions, gut inflammation was associated with higher intestinal cholesterol and cholic acid concentrations and a reduced microbial diversity. The latter was accompanied by a proliferation of Robinsoniella peoriensis, Clostridium innocuum, Escherichia coli, and Enterococcus gallinarum. All these species proved to be highly bile acid resistant. We concluded that chronic colitis in IL-10-/- mice is associated with changes in intestinal steroid profiles. These changes may be due to alterations in gut microbiota composition or vice versa. Whether the bacterial sterol and bile acid metabolism is implicated in colitis and colorectal carcinoma etiology remains to be clarified.

Influence of in vitro supplementation with lipids from conventional and Alpine milk on fatty acid distribution and cell growth of HT-29 cells.

BACKGROUND: To date, the influence of milk and dairy products on carcinogenesis remains controversial. However, lipids of ruminant origin such as conjugated linoleic acids (CLA) are known to exhibit beneficial effects in vitro and in vivo. The aim of the present study was to determine the influence of milk lipids of different origin and varying quality presenting as free fatty acid (FFA) solutions on cellular fatty acid distribution, cellular viability, and growth of human colon adenocarcinoma cells (HT-29). METHODS: FAME of conventional and Alpine milk lipids (MLcon, MLalp) and cells treated with FFA derivatives of milk lipids were analyzed by means of GC-FID and Ag+-HPLC. Cellular viability and growth of the cells were determined by means of CellTiter-Blue®-assay and DAPI-assay (4',6-diamidino-2-phenylindole dihydrochloride), respectively. RESULTS: Supplementation with milk lipids significantly decreased viability and growth of HT-29 cells in a dose- and time-dependent manner. MLalp showed a lower SFA/MUFA ratio, a 8 fold increased CLA content, and different CLA profile compared to MLcon but did not demonstrate additional growth-inhibitory effects. In addition, total concentration and fatty acid distribution of cellular lipids were altered. In particular, treatment of the cells yielded highest amounts of two types of milk specific major fatty acids (µg FA/mg cellular protein) after 8 h of incubation compared to 24 h; 200 µM of MLcon (C16:0, 206 ± 43), 200 µM of MLalp (C18:1 c9, (223 ± 19). Vaccenic acid (C18:1 t11) contained in milk lipids was converted to c9,t11-CLA in HT-29 cells. Notably, the ratio of t11,c13-CLA/t7,c9-CLA, a criterion for pasture feeding of the cows, was significantly changed after incubation for 8 h with lipids from MLalp (3.6 - 4.8), compared to lipids from MLcon (0.3 - 0.6). CONCLUSIONS: Natural lipids from conventional and Alpine milk showed similar growth inhibitory effects. However, different changes in cellular lipid composition suggested a milk lipid-depending influence on cell sensitivity. It is expected that similar changes may also be evident in other cell lines. To our knowledge, this is the first study showing a varied impact of complex milk lipids on fatty acid distribution in a colon cancer cell line.

Increased plasma plant sterol concentrations and a heterozygous amino acid exchange in ATP binding cassette transporter ABCG5: a case report.

Whilst conducting a scientific study, an elevated plasma plant sterol concentration of 3.07 mg/dL was established in one proband. Similar levels found in his mothers plasma (2.73 mg/dL) were suggestive of a heterozygous sitosterolemia. The resulting gene analysis for ATP binding cassette transporter G5/G8 (ABCG5/G8) revealed a heterozygous polymorphism in ABCG8 (Thr400Lys, rs4148217), which the proband had inherited from his father. However, a heterozygous amino acid exchange (Arg406Gln) in exon 9 of ABCG5 was revealed, which was inherited from his mother. Although not sufficient evidence exists to regard this sequence variation as a mutation, this previously unreleased sequence variation occurred in a "hot spot" area for sitosterolemia of the ABCG5 gene (exon 9) and the similar increased plasma plant sterol concentrations of the heterozygous mother contribute to the notion, that this very likely presents an inactivating mutation.

Work and diet-related risk factors of cardiovascular diseases: comparison of two occupational groups.

BACKGROUND: Although work related risk factors associated with Cardiovascular Diseases (CD) have been well researched, there is no detailed knowledge regarding disparate occupational groups each with a different risk exposition. Therefore, two occupational groups (chefs and office workers) were compared with a focus on nutritional and psychosocial factors. METHODS: Two groups of subjects were tested for work and diet-related risks of CD (45 chefs and 48 office workers). The groups matched both for gender (male) and age (30 to 45 years). The study included a medical check-up, bioelectrical impedance analysis as well as an evaluation of questionnaires on health, nutritional behaviour and coping capacity. In addition, volunteers were required to compile a 7-day-dietary-record and collect their urine 24 h prior to their check-up. Blood samples drawn were analysed for glucose and lipid metabolism, homocysteine, vitamin B12, folic acid; C-reactive protein, uric acid, red blood cell fatty acids, plant sterols, antioxidative capacity and oxidative stress. RESULTS: On average, the chefs showed one risk factor more compared to the office workers. The most frequent risk factors in both groups included overweight/obesity (chef group [CG]: 62.2%; office group [OG]: 58.3%) and elevated TC (CG: 62.2%; OG: 43.8%]. Moreover, although the chefs often had higher CRP-concentrations (40.0%), more office workers suffered from hypertension (37.5%).Chefs showed significant higher concentrations of saturated fatty acids and oleic acid, whereas docosahexaenoic acid, Omega-6- and trans fatty acids were found more frequently in the red blood cell membranes of office workers. While there were no significant differences in analysed plant sterols between the two occupational groups, 7,8-dihydro-8-oxo-2'-deoxyguanosine was significantly increased in office workers.Concerning the work-related psychosocial factors, the chefs were characterised by a stronger subjective importance of work, a greater degree of professional aspiration and enhanced efforts at perfectionism at their workplace. CONCLUSIONS: The chefs in the study bear a higher risk of CD compared to the office-workers. Although, CD is not exclusively a result of workplace-conditions, study results show that work-related influences can not be ignored. Thus, prevention of CD may be an important task attributable to occupational physicians.

Faecal steroid excretion in humans is affected by calcium supplementation and shows gender-specific differences.

BACKGROUND: Previous human studies on the effect of dietary calcium supplementation on faecal excretion of bile acids (BA) and faecal water concentrations of animal neutral sterols (NSt, cholesterol and its metabolites) lack detailed information about single BA and NSt. AIM OF THE STUDY: We investigated whether single BA and NSt in faeces and especially in faecal water are affected by calcium supplementation and whether this affects genotoxicity of faecal water. In addition, we differentiated between men and women with regard to the concentrations of BA and NSt in faecal water. METHODS: Thirty-one healthy volunteers consumed a calcium supplemented bread (1.0 g/day) and a placebo bread, respectively, for 4 weeks in a double-blind, randomised cross-over trial. Faeces were collected quantitatively for 5 days in the last week of each period. NSt and BA were analysed by GC-MS. RESULTS: Due to calcium supplementation faecal concentrations of lithocholic acid (LCA, 14%, P = 0.008), deoxycholic acid (DCA, 19%, P < 0.001) and 12 keto-deoxycholic acid (12 keto DCA, 29%, P = 0.049) significantly increased whereas BA concentrations in faecal water were only marginally affected. In contrast, concentrations of cholesterol (30%, P = 0.020) and its metabolites coprostanol (43%, P = 0.004), coprostanone (36%, P = 0.003), cholestanol (44%, P = 0.001) and cholestenone (32%, P = 0.038) in faecal water significantly decreased. Total NSt concentration in faecal water was found to be significantly higher in women compared to men (P = 0.018). The genotoxicity of faecal water was neither affected by calcium supplementation nor were there gender-specific differences. CONCLUSIONS: Dietary calcium supplementation diversely affects BA and NSt in faeces and in faecal water but does not influence the genotoxicity of faecal water in healthy adults.

Octacosanol administration to humans decreases neutral sterol and bile acid concentration in feces.

To investigate octacosanol (OC) metabolism in humans and its influence on cholesterol metabolism, two studies were conducted. In the first study ten healthy women received daily 30 mg OC for a period of 4 weeks. Blood and feces samples were collected at baseline and after the intervention. Serum concentrations of total cholesterol, LDL cholesterol, and HDL cholesterol were not altered following OC administration. Concentrations of excreted cholesterol end products decreased with the intervention (neutral sterols: 24.6 +/- 9.7 mg/g vs. 20.3 +/- 7.5 mg/g dry matter, P < 0.05; bile acids: 6.47 +/- 3.89 mg/g vs. 4.03 +/- 2.26 mg/g dry matter, P < 0.05). OC was not detected in serum samples, but the fecal OC concentration increased after the intervention period (11 +/- 7 microg/g vs. 817 +/- 179 microg/g dry matter, P < 0.05). In the second kinetic study on three participants, OC was identified in serums after oral application of 50 mg OC within 8 h. The decrease in the concentration of fecal cholesterol end products may underline a systemic effect of OC on cholesterol metabolism, even though the serum cholesterol levels were not influenced.

Nanoscale and customary non-esterified sitosterols are equally enriched in different body compartments of the guinea pig.

The impact of sitosterol formulation particle size on the intestinal sterol absorption and the sterol status in various tissues in Dunkin Hartley guinea pigs was investigated. Three groups of animals (six each) were fed a basal diet ("control") or a basal diet containing either customary sitosterol ("customary", particle size: 10 000-90 000 nm) or nanoscale sitosterol ("nanoscale", particle size: 200-300 nm). The average daily sitosterol intake was 21 +/- 7 mg (control), 154 +/- 8 mg (customary), and 127 +/- 18 mg (nanoscale) for 2 weeks. Sitosterol and cholesterol were analyzed in samples of plasma, blood cells, bile, liver, kidney, jejunal mucosa/serosa, cecum, colon and feces. Concentrations of sitosterol in all analyzed matrices increased significantly in the supplemented groups when compared to control group. No differences in the sitosterol concentrations in analyzed matrices occurred between nanoscale and customary group. The cholesterol concentrations in tissues remained unchanged. Fecal fatty acid and sterol distributions were modified during sitosterol intervention. Both particle sizes equally increased sitosterol levels in cholesterol-metabolizing compartments in the guinea pig. No differences in body compartment accumulation and intestinal absorption of the different sitosterol particle sizes were observed.

Prevention of colitis-associated carcinogenesis in a mouse model by diet supplementation with ursodeoxycholic acid.

Bile acids in the intestinal lumen contribute to the homeostatic regulation of proliferation and death of the colonic epithelial cells: Deoxycholic acid (DCA) appears to enhance and ursodeoxycholic acid (UDCA) to attenuate the process of chemically induced carcinogenesis. We studied the effects of UDCA on colitis-related colorectal carcinogenesis. Three groups of 25 mice were given 0.7% dextran sulphate in drinking water for 7 days and pure water for 10 days and were fed a standard diet containing double iron concentration. In 2 groups, the diet was supplemented with 0.2% cholic acid (CA), the precursor of DCA, or with 0.4% UDCA. After 15 cycles, the histology, the expression of MUC2, beta-catenin, p27 and p16 and the fecal water concentration of DCA and UDCA were investigated. All animals showed colitis with similar severity and histologic as well as immunophenotypic alterations, resembling those of human colitis. Among the animals fed the nonsupplemented diet, 46% developed colorectal adenocarcinomas and 54% anal-rectal squamous cell carcinomas. The prevalence of dysplasia and carcinomas did not change significantly in the animals given CA. Among the mice fed with UDCA, none developed adenocarcinomas and 20% squamous carcinomas. Dysplastic lesions were found in 88%, 67% and 40% of each group, respectively. The prevalence of dysplasia as well as of carcinoma showed an inverse relationship to the UDCA concentration in the fecal water. These data indicate that UDCA suppresses colitis-associated carcinogenesis. This model is suitable for investigation of the mechanism of the anticarcinogenic effect of UDCA in vivo.

Cholesterol metabolism is affected by calcium phosphate supplementation in humans.

Dietary calcium and phosphate precipitate in the small intestine to form insoluble amorphous calcium phosphate (ACP). The ability of ACP to bind and inactivate luminal bile acids might have an effect on cholesterol metabolism. To test this hypothesis, a placebo-controlled, double-blind, crossover study with pentacalcium hydroxy-triphosphate supplementation (CaP; 1.0 g elemental calcium) was conducted in 31 young healthy volunteers. The CaP was incorporated into bread. Serum cholesterol concentrations were lower after 4 wk of supplementation than after 4 wk of placebo (4.36 vs. 4.60 mmol/L; P = 0.008). Serum LDL cholesterol and the ratio of LDL:HDL cholesterol also tended to be lower after CaP supplementation than after placebo (-5.6%, P = 0.083 and -5.4%, P < 0.062, respectively). The participants' fat and cholesterol intakes and fecal fat excretion did not differ in the 2 periods. Although the analysis of fecal samples showed no difference in the excretion of total neutral sterols (sum of cholesterol and its transformation products), the excretion of cholesterol itself increased (9.64 vs. 5.80 micromol/g dry matter; P = 0.025; n = 25), whereas the excretion of the metabolite coprostanol decreased (18.5 vs. 21.0 mumol/g dry matter; P = 0.025; n = 25) in the CaP period. Bile acid excretion increased during the CaP period compared with the placebo period (25.4 vs. 22.9 micromol/g dry matter; P = 0.003). The observed beneficial effects on cholesterol metabolism are not the result of an increased excretion of cholesterol, but might be explained by an increased bile acid excretion and a subsequent regeneration of bile acids from endogenous cholesterol in the liver.

Determination of underivatised sterols and bile acid trimethyl silyl ether methyl esters by gas chromatography-mass spectrometry-single ion monitoring in faeces.

A method for quantification of total faecal sterols and bile acids (BAs) in human stool by using gas chromatography-mass spectrometry-single ion monitoring (GC-MS-SIM) is described. Cholesterol, coprostanol, coprostanone, cholestanol, iso-lithocholic acid (iso-LCA), lithocholic acid (LCA), iso-deoxycholic acid (iso-DCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), cholic acid (CA), and 12-oxo-deoxycholic acid (12-oxo-DCA) in faeces of 86 healthy subjects were determined. The sample preparation for sterol analysis requires hydrolysis and liquid extraction from matrix, but no derivatisation. The GC-flame ionisation detection (FID) and total ion current (TIC) in GC-MS were not sufficient for sterol and BA determination, whereas selectivity and specificity of the GC-MS-SIM ensured the analysis of sterols and BAs in faeces.