Extraction Of Bile Acids Research Articles

Quantification and identification of bile acids in saliva by liquid chromatography-mass spectrometry: Possible non-invasive diagnostics of Barrett's esophagus?

Bile acids are a group of steroid compounds essential for lipid digestion. However, when bile acids are refluxed into the stomach and the esophagus, during the so called duodenogastroesophageal reflux, they can have a detrimental effect on the esophageal epithelium and cause pathological changes of esophageal tissue, e.g., Barrett’s esophagus (BE). The levels of bile acids in saliva could therefore serve as possible biomarkers for the diagnostics of BE. In this work, we focused on optimization of sample collection and preparation by solid-phase extraction and subsequent quantification of 11 bile acids (unconjugated, glycine-conjugated) in saliva from healthy volunteers and BE patients by ultra-high-performance liquid chromatography coupled to triple-quadrupole tandem mass spectrometry. Moreover, high resolution MS (Orbitrap-MS) was utilized for identification of new bile acids in saliva. Methods for saliva collection including simple spitting and the Salivette® saliva collection system were compared; the latter was found to be unsuitable due to excessive retention of bile acids in the cotton swab. Methanol with 0.1% formic acid were selected for protein precipitation and bile acid extraction prior to SPE. Separation was performed in gradient elution of methanol and 0.1% formic acid in less than 10 min. Saliva from BE patients contained higher levels of almost all bile acids, and the tested groups could be distinguished by principal component analysis. In untargeted analysis by high resolution MS, taurine-conjugated bile acids and glycine-conjugated dihydroxy-bile acid sulfate were identified in saliva from healthy volunteers. We propose that analysis of salivary bile acids including taurine conjugates could be applicable in diagnostics of BE, following a larger clinical study.

One-Pot Extraction and Quantification Method for Bile Acids in the Rat Liver by Capillary Liquid Chromatography Tandem Mass Spectrometry.

We developed a highly sensitive method for quantifying 21 bile acids (BAs) in the rat liver by capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS) with one-pot extraction. High recovery rates were obtained for the one-pot methods with either methanol (MeOH) extraction or MeOH/acetonitrile (ACN) (1:1, v/v) mixture extraction; the results obtained for the MeOH/ACN mixture solution were better than the results obtained for MeOH. Thus, we determined that the one-pot method with MeOH/ACN was the most suitable method for the efficient extraction of BAs in the liver. Targeted BAs were well separated by cLC with gradient elution using ammonium acetate (NH4OAc)–MeOH mobile phases. Method validation proved that the intra-day and inter-day accuracies and precisions were primarily less than ±20 and 20% relative standard deviation, respectively. Also, the limit of detection (LOD) and the limit of quantitation (LOQ) were 0.9–10 and 2.3–27 ng/g liver, which proves the high sensitivity of the method. Finally, we quantitated 21 BA concentrations in the liver samples of normal and nonalcoholic steatohepatitis (NASH) rats, both of which were derived from stroke-prone spontaneously hypertensive five (SHRSP5) /Dmcr rat. The hepatic BA profiles were found to be substantially different between the normal and NASH groups; the two groups were clearly separated along the first component axis in the score plots of the principal component analysis. In particular, 10 BAs (β-muricholic acid (MCA), glyco (G-) cholic acid (CA), G-chenodeoxycholic acid (CDCA), tauro (T-) CA, T-CDCA, T-ursodeoxycholic acid (UDCA), T-lithocholic acid (LCA), T-hiodeoxycholic acid (HDCA), T-α-MCA, and T-β-MCA) were significantly different between the two groups using Welch’s t-test with the false discovery rate correction method, demonstrating BA disruption in the NASH model rat. In conclusion, this method was able to quantify 21 BAs in the rat liver and will evaluate the hepatic BA pathophysiology of rat disease models.

Extraction and quantitative determination of bile acids in feces

With rapid advances in gut microbiome research, fecal bile acids are increasingly being monitored as potential biomarkers of diet related disease susceptibility. As such, rapid, robust and reliable methods for their analysis are of increasing importance. Herein is described a simple extraction method for the analysis of bile acids in feces suitable for subsequent quantification by liquid chromatography and tandem mass spectrometry. A C18 column separated the analytes with excellent peak shape and retention time repeatability maintained across 800 injections. The intra-day and inter-day precision and accuracy was greater than 80%. Recoveries ranged from 83.58 to 122.41%. The limit of detection and limit of quantification were in the range 2.5–15 nM, respectively. The optimized method involved extracting bile acids from wet feces with minimal clean up. A second aliquot of fecal material was dried and weighed to correct for water content. Extracting from dried feces showed reduced recovery that could be corrected for by spiking the feces with deuterated standards prior to drying. Storage of the extracts and standards in a refrigerated autosampler prior to analysis on the LC-MS is necessary. Multiple freeze-thaws of both extracts and standards lead to poor recoveries for some bile acids. The method was successfully applied to 100 human fecal samples.

Preparation of magnetic mesoporous epoxy resin by initiator-free ring-opening polymerization for extraction of bile acids from human serum

In this work, we reported a simple two-step method for the synthesis of magnetic mesoporous epoxy resin (MMER), including one-pot template-free hydrothermal synthesis of nanoscale amine-functionalized magnetic nanoparticles (MN-NH2) and initiator-free ring-opening polymerization of epoxy resin. The resultant MMER was characterized in detail by transmission electron microscope (TEM), Fourier transform-infrared (FT-IR) spectra, X-ray photoelectron spectroscopy (XPS), thermogravimetic analysis (TGA) and magnetization curves. These results demonstrated successful synthesis of MMER with sufficient magnetic property and excellent thermal stability. The epoxy resin was covalent bonding MN-NH2 on and synthesized by hydrophobic monomers, so the MMER exhibited excellent adsorption quantity for hydrophobic bile acids. The MMER was used as magnetic solid-phase extraction (MSPE) sorbent, and combined with liquid chromatography-tandem mass spectrometry to extract and monitor 11 kinds of bile acids from serum sample. The proposed MSPE combined with LC–MS/MS method exhibited low limit of detection between 0.1 and 5 ng mL−1. In blank serum sample, 9 kinds of bile acids were detected, and ranged from −2.29 ng mL−1 to 6.86 ng mL−1. In standard addition recovery test, the recovery values of detectable bile acids ranged 102.4% to 108.5%, 96.0% to 104.0% and 82.3% to 103.3% when spiked with 0.2, 2.0 and 20 ng mL−1, respectively. The intra- and inter-day precision (n = 6) ranged 3.7% to 5.9% and 7.0% to 9.5%, respectively. The above results demonstrated that the MSPE combined with LC–MS/MS method was accurate and effective for quantitative determination of bile acids from complex biological samples.

High-Efficiency, Matrix Interference-Free, General Applicable Probes for Bile Acids Extraction and Detection.

Although bile acids (BAs) have been suggested as important biomarkers for endocrine diseases, the identification and quantification of different BAs are still challenges due to their enormous species and wide range concentrations. Herein, a copolymer probe based on β‐cyclodextrin (β‐CD) is fabricated through a simple in‐mold photopolymerization for the selective extraction of BAs. Through the unique stereochemical affinity between BAs and the cavity of β‐CD, the custom probe shows superior enriching capacities to series BAs. Moreover, the outstanding extraction ability is proved to be consistent in various interfering conditions, including pH changing and the addition of complex matrix. Further comparison shows that the stereostructure of the nucleus of BAs plays a vital role during the formation of the β‐CD/BA complex, indicating the potential for efficient extraction of other BAs, including their structural analogues or some unknown ones. The developed probe is used for solid phase microextraction, and the limits of detection are lower than 0.075 ng mL−1 by coupling to high performance liquid chromatography‐tandem mass analysis. The results in this study highlight the potential for effective improvement of immediate detection and profiling of BAs in real samples, which will make a tremendous impact in the analytical field or clinical diagnosis.

A technique for extraction and Thin Layer Chromatography visualization of fecal bile acids applied to neotropical felid scats

Fecal bile acid patterns have been used successfully to identify scats. Neotropical felid scats are capable of this biochemical identification because they present low concentrations of plant pigments that would interfere in fecal bile acids detection. However, neotropical felid scats have poor quantities of bile acids, so we developed in this work a proper technique for their extraction, visualization and determination. Twenty eighth feces of seven different felid species, collected from Zoological and Wildlife Parks, were dried and pulverized. The procedure for analyzing feces is: Take one g of pulverized feces and shake for 3 hr at room temperature in 20 ml benzene:methanol; filter and evaporate to 5 ml. Spot on TLC plate and develop in toluene:acetic acid:water. Dry and visualize with anisaldehyde. Field collected scats could be identified by the bile acids pattern revealed by this specific technique and, then, used as a source of information for distribution, density and food habits studies.

Profiling of urinary bile acids in piglets by a combination of enzymatic deconjugation and targeted LC-MRM-MS

We present a method using a combination of enzymatic deconjugation and targeted LC-multiple reaction monitoring (MRM)-MS analysis for analyzing all common bile acids (BAs) in piglet urine, and in particular, for detecting conjugated BAs either in the absence of their standards, or when present in low concentrations. Initially, before enzymatic deconjugation, 19 unconjugated BAs (FBAs) were detected where the total concentration of the detected FBAs was 9.90 μmol/l. Sixty-seven conjugated BAs were identified by LC-MRM-MS analysis before and after enzymatic deconjugation. Four enzymatic assays were used to deconjugate the BA conjugates. FBAs in urine after cholylglycine hydrolase/sulfatase treatment were 33.40 μmol/l, indicating the urinary BAs were comprised of 29.75% FBAs and 70.25% conjugated BAs in single and multiple conjugated forms. For the conjugates in single form, released FBAs from cholylglycine hydrolase deconjugation indicated that the conjugates with amino acids were 14.54% of urinary BAs, 16.27% glycosidic conjugates were found by β-glucuronidase treatment, and sulfatase with glucuronidase inhibitor treatment liberated FBAs that constituted 16.67% of urinary BAs. Notably, chenodeoxycholic acid (CDCA) was initially detected only in trace amounts in urine, but was found at significant levels after the enzymatic assays above. These results support that CDCA is a precursor of γ-muricholic acid in BA biosynthesis in piglets.

Iron Oxide Magnetic Nanoparticles Mediated Extraction of Toxic Bile Acids Form Inexpensive Nutrient Media for Unprecedented Growth Enhancement of Halophilic Bacteria

Iron Oxide Magnetic Nanoparticles Mediated Extraction of Toxic Bile Acids Form Inexpensive Nutrient Media for Unprecedented Growth Enhancement of Halophilic Bacteria

New method for the determination of bile acids in human plasma by liquid-phase microextraction using liquid chromatography-ion-trap-time-of-flight mass spectrometry

Bile acids (BAs) are derived from cholesterol and produced in the liver. The most abundant bile acids in humans are usually conjugated with glycine and taurine and are divided into primary BAs such as cholic acid (CA) and chenodeoxycholic acid (CDCA) and secondary BAs like deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA).The differences amongst individual bile acids (BAs) are significant in order to distinguish different pathological processes and exposure to chemical compounds. Hollow fiber based liquid-phase microextraction (HF-LPME) is a technique that combines sample cleansing, extraction and the concentration of analytes, where a hydrophobic porous capillary membrane is impregnated with an organic extraction solvent and the lumen is filled with microliters of a phase acceptor both organic by nature. The aim of this study was to develop a new method to extract bile acids from plasma through HF-LPME of two phases (octanol as the acceptor phase) using LCMS-IT-TOF. The optimized two-phased LPME procedure for the extraction of bile acids showed limits of detection 1.0μgL−1 and limits of quantification of 5.0μgL−1. The intra-assay precision ranged from 2.1 to 11.9%. The method developed was linear over the range of 5.0–200.0μgL−1 for all analytes. The hollow-fiber liquid-phase microextraction method was applied to human plasma from workers exposed to organic and halogenated solvents and also to unexposed volunteers. The method is simple, low cost and it does not require large amounts of organic solvents, therefore it is quite suitable for the analysis of bile acids exposed to hepatotoxic compounds.

Dyslipidemic and Antioxidant activity ofPiper longumextract in hyperlipemic rats

The dyslipidemic and antioxidant activity of Piper longum extract has been studied in triton and high fat diet fed hyperlipemic rats. The extract lowered the serum lipids at (200 mg/kg, b.w., p.o.) in triton WR-1339 induced hyperlipidemia. Chronic feeding of this extract (100 mg/kg, b.w.) in animals simultaneously fed with high fat diet (HFD) for 30 days caused lowering of lipid and apoproteins level of lipoproteins in experimental animals. P. langum activates lipolytic enzymes in plasma and liver lipids. These extract increased faecal bile acid extraction and enhanced plasma lecithin: cholesterol acyltransferase activity. Piper langum extract showed potent inhibition of antioxidant and scavenger of oxygen free radical activity (in vitro) and it induced oxidative stress.

A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis.

Determination of bile acid profiles in scat samples of wild animals by liquid chromatography-electrospray mass spectrometry

A practical and accurate analytical method using liquid chromatography-mass spectrometry (LC-MS) was developed to determine the bile acid profile in the scat samples of various wild animals such as mountain lion, bobcat, coyote, raccoon, fox, etc. Bile acid extraction from the scat sample of the wild animals and its detection using the LC-MS technique are very easy and can be done in a short period of time with good resolution. The bile acid profiles are different in each species and the analytical method is very effective in identifying the wild animals under investigation. The detection limits of all bile acids were very low (∼1.0 ng mL−1) and the analytical method provides excellent reproducibility.

Bile acid profiling in human biological samples: Comparison of extraction procedures and application to normal and cholestatic patients

The role of bile acids in cell metabolism, membrane biology and cell signaling is increasingly recognized, thus making necessary a robust and versatile technique to extract, separate and quantify a large concentration range of these numerous molecular species. HPLC–MS/MS analysis provides the highest sensitivity to detect and identify bile acids. However, due to their large chemical diversity, extraction methods are critical and quite difficult to optimize, as shown by a survey of the literature. This paper compares the performances of four bile acid extraction protocols applied to either liquid (serum, urine, bile) or solid (stool) samples. Acetonitrile was found to be the best solvent for deproteinizing liquid samples and NaOH the best one for stool extraction. These optimized extraction procedures allowed us to quantitate as much as 27 distinct bile acids including sulfated species in a unique 30min HPLC run, including both hydrophilic and hydrophobic species with a high efficiency. Tandem MS provided a non ambiguous identification of each metabolite with a good sensitivity (LOQ below 20nmol/l except for THDCA and TLCA). After validation, these methods, successfully applied to a group of 39 control patients, detected 14 different species in serum in the range of 30–800nmol/l, 11 species in urine in the range of 20–200nmol/l and 25 species in stool in the range of 0.4–2000nmol/g. The clinical interest of this method has been then validated on cholestatic patients. The proposed protocols seem suitable for profiling bile acids in routine analysis.

Rapid quantification of conjugated and unconjugated bile acids and C27 precursors in dried blood spots and small volumes of serum

The aim of the study was to develop a method for fast and reliable diagnosis of peroxisomal diseases and to facilitate differential diagnosis of cholestatic hepatopathy. For the quantification of bile acids and their conjugates as well as C(27) precursors di- and trihydroxycholestanoic acid (DHCA, THCA), in small pediatric blood samples we combined HPLC separation on a reverse-phase C18 column with ESI-MS/MS analysis in the negative ion mode. Analysis was done with good precision (CV 3,7%-11.1%) and sufficient sensitivity (LOQ: 11-91 nmol/L) without derivatization. Complete analysis of 17 free and conjugated bile acids from dried blood spots and 10 microL serum samples, respectively, was performed within 12 min. Measurement of conjugated primary bile acids plus DHCA and THCA as well as ursodeoxycholic acid was done in 4.5 min. In blood spots of healthy newborns, conjugated primary bile acids were found in the range of 0.01 to 2.01 micromol/L. Concentrations of C(27) precursors were below the detection limit in normal controls. DHCA and THCA were specifically elevated in cases of peroxysomal defects and one Zellweger patient.

A Prospero-related Homeodomain Protein Is a Novel Co-regulator of Hepatocyte Nuclear Factor 4α That Regulates the Cholesterol 7α-Hydroxylase Gene

Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with alpha-fetoprotein transcription factor and repress cholesterol 7alpha-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4alpha, an important transactivator of the human CYP7A1 gene in bile acid synthesis and phosphoenolpyruvate carboxykinase (PEPCK) gene in gluconeogenesis. A real time PCR assay detected Prox1 mRNA expression in human primary hepatocytes and HepG2 cells. Reporter assay, GST pull-down, co-immunoprecipitation, and yeast two-hybrid assays identified a specific interaction between the N-terminal LXXLL motif of Prox1 and the activation function 2 domain of HNF4alpha. Prox1 strongly inhibited HNF4alpha and peroxisome proliferators-activated receptor gamma coactivator-1alpha co-activation of the CYP7A1 and PEPCK genes. Knock down of the endogenous Prox1 by small interfering RNA resulted in significant increase of CYP7A1 and PEPCK mRNA expression and the rate of bile acid synthesis in HepG2 cells. These results suggest that Prox1 is a novel co-regulator of HNF4alpha that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver.

Unconjugated faecal bile acids in familial adenomatous polyposis analysed by gas-liquid chromatography and mass spectrometry.

Previous studies have suggested reduced formation of secondary bile acids in patients with familial adenomatous polyposis (FAP). Developments in the collection, extraction and analysis of faecal bile acids as well as in the accurate diagnosis of FAP by DNA markers prompted reinvestigation of this hypothesis. The median (interquartile range (i.q.r.)) faecal bile acid concentration (3.69 (1.66-5.36) mumol per g dry weight) and daily excretion rate (60.5 (29-149) mumol per g per 24 h) in ten patients with FAP were similar to those of nine control subjects (3.31 (0.65-8.38) mumol per g dry weight and 30.1 (7.9-228) mumol per g per 24 h). Although the median (i.q.r.) concentration of only one bile acid (12-oxo-lithocholic acid) was significantly different between patients with FAP and controls (49 (34-70) versus 0 (0-20) nmol per g dry weight, P = 0.006), the derivatives of chenodeoxycholic acid (3.35 (1.76-5.32) versus 0.51 (0.13-2.37) mumol per g dry weight, P = 0.02) and cholic acid (1.63 (0.42-2.34) versus 0.80 (0.13-3.57) mumol per g dry weight, P = 0.006) were increased in those with polyposis. These results show increased bacterial biotransformation of faecal bile acids in patients with FAP.

Metabolic profiles and bile acid extraction rate in the liver of cows with fasting-induced hepatic lipidosis.

This study was designed to monitor lipid profile in the portal and hepatic blood of cows with fasting-induced hepatic lipidosis, and to compare the results with those in the jugular blood. The work was also carried out to investigate bile acid (BA) in these vessels, and further to investigate BA extraction rate in the liver. Five cows were equipped with catheters in the portal, hepatic and jugular veins (day 0), fasted for 4 days (day 1-day 4) and then refed (day 5-day 11). Before morning feeding, blood was sampled before, during and after fasting from the catheterized vessels. In the portal blood, the concentration of non-esterified fatty acids (NEFA) showed a progressive increase and at day 5 there was an approximate twofold rise. Increased NEFA concentrations were also found similarly in the other two veins. At day 5, beta-hydroxybutyrate (BHBA) in the portal, hepatic and jugular blood rose to 197, 190 and 186% of the pre-fasting value, respectively. However, the concentrations of NEFA and BHBA in the three veins gradually returned to pre-fasting concentration during the refeeding period. Compared with the pre-fasting value at day 0, the content of liver triglyceride (TG) increased significantly at day 5 (P < 0.01). In the liver, the hepatic extraction rate of BA dropped from 3.1 times pre-fasting to 2.2 times during fasting. There were no significant differences in the concentrations of glucose, TG, total cholesterol, cholesterol esters, free cholesterol and phospholipids. The results of the current study show that metabolic alterations occur in the portal, hepatic and jugular veins during induction of hepatic lipidosis in cows, and mostly metabolites, with exception of BA concentration, run parallel. The decreased BA extraction rate in the liver of fasted cows was considered to reflect hepatic cell impairment caused by TG accumulation. Hopefully, the findings, at least in part, contribute to the explanation of the pathophysiology of hepatic lipidosis in dairy cows.

Hepatocyte Nuclear Factor 4α Is a Central Regulator of Bile Acid Conjugation

Hepatocyte nuclear factor 4alpha (HNF4alpha) has an important role in regulating the expression of liver-specific genes. Because bile acids are produced from cholesterol in liver and many enzymes involved in their biosynthesis are preferentially expressed in liver, the role of HNF4alpha in the regulation of bile acid production was examined. In mice, unconjugated bile acids are conjugated with taurine by the liver-specific enzymes, bile acid-CoA ligase and bile acid-CoA:amino acid N-acyltransferase (BAT). Mice lacking hepatic HNF4alpha expression exhibited markedly decreased expression of the very long chain acyl-CoA synthase-related gene (VLACSR), a mouse candidate for bile acid-CoA ligase, and BAT. This was associated with markedly elevated levels of unconjugated and glycine-conjugated bile acids in gallbladder. HNF4alpha was found to bind directly to the mouse VLACSR and BAT gene promoters, and the promoter activities were dependent on HNF4alpha-binding sites and HNF4alpha expression. In conclusion, HNF4alpha plays a central role in bile acid conjugation by direct regulation of VLACSR and BAT in vivo.

Simultaneous quantitation of fatty acids, sterols and bile acids in human stool by capillary gas–liquid chromatography

A simple method for the simultaneous gas–liquid chromatographic quantitation of fatty acids, sterols and bile acids from human fecal samples is described. The various compounds are directly converted into the n-butyl ester-trimethylsilyl ether derivatives, without prior isolation from the stool. Under these conditions, fecal bile acid derivatives are well resolved from each other and from those of fecal fatty acids and sterols without overlaps. The method was found to be reproducible and recoveries were similar to those obtained after exhaustive solvent extraction of fecal sterols, fatty acids and bile acids. Optimum derivatization conditions that allowed maximum recovery of fecal components with minimal destruction and application of the method for simultaneous bile acid, fatty acid and sterol analysis in human stool are described.

The Diet1 Locus Confers Protection against Hypercholesterolemia through Enhanced Bile Acid Metabolism

The C57BL/6ByJ (B6By) mouse strain is resistant to diet-induced hypercholesterolemia and atherosclerosis, despite its near genetic identity with the atherosclerosis-susceptible C57BL/6J (B6J) strain. We previously identified a genetic locus, Diet1, which is responsible for the resistant phenotype in B6By mice. To investigate the function of Diet1, we compared mRNA expression profiles in the liver of B6By and B6J mice fed an atherogenic diet using a DNA microarray. These studies revealed elevated expression levels in B6By liver for key bile acid synthesis proteins, including cholesterol 7alpha-hydroxylase and sterol-27-hydroxylase, and the oxysterol nuclear receptor liver X receptor alpha. Expression levels for several other genes involved in bile acid metabolism were subsequently found to differ between B6By and B6J mice, including the bile acid receptor farnesoid X receptor, oxysterol 7alpha-hydroxylase, sterol-12alpha-hydroxylase, and hepatic bile acid transporters on both sinusoidal and canalicular membranes. The overall expression profile of the B6By strain suggests a higher rate of bile acid synthesis and transport in these mice. Consistent with this interpretation, fecal bile acid excretion is increased 2-fold in B6By mice, and bile acid levels in blood and urine are elevated 3- and 18-fold, respectively. Genetic analysis of serum bile acid levels revealed co-segregation with Diet1, indicating that this locus is likely responsible for both increased bile acid excretion and resistance to hypercholesterolemia in B6By mice.