Quantitation of free and conjugated bile acids in human feces using a high-pressure liquid chromatography counterion method

A method has been developed for extraction and purification of the major bile acids in human feces, and for their quantitative estimation using high-pressure liquid chromatography. Freeze-dried fecal material was extracted with alkaline ethanol and, after removal of neutral steroids, was subjected to thin-layer chromatography, followed by reversed-phase C18 silica cartridge (Sep-Pak) purification. The mixture was further separated into free, and glyco and tauro conjugates by ion-exchange chromatography. Subsequent resolution of individual bile acids was accomplished by HPLC using a counterion pairing method.     

Determination of fetal bile acids in biological fluids from neonates by gas chromatography-negative ion chemical ionization mass spectrometry

A method has been developed for microanalysis of fetal bile acids in biological fluids from neonates by capillary gas chromatography-mass spectrometry using negative-ion chemical ionization of pentafluorobenzyl ester-dimethylethylsilyl ether derivatives of bile acids. Calibration curves for the bile acid derivatives are useful over the range 0.1–100 pg and the detection limit for bile acids was 1 fg (S/N=5) using isobutane as a reagent gas. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free serum and dried blood discs ranged from 92 to 101% and from 93 to 108%, respectively, of the added amounts of their standard samples. The analysis of bile acids on a dried blood disc, meconium and urine from infants, exhibited significant hydroxylation at the 1β-, 2β-, 4β and 6α-positions of the usual bile acids, cholic and chenodeoxycholic acids, for the urinary or fecal excretion of bile acids in the fetal and neonatal periods. The present method was applied clinically to analyze bile acids on a dried blood disc from neonatal patients with congenital biliary atresia and hyper-bile-acidemia.     

Separation and detection of uv-absorbing derivatives of fecal bile acid metabolites by high-performance liquid chromatography

Major fecal bile acid metabolites related to lithocholic acid were resolved by high-performance liquid chromatography (hplc). The uv-absorbing p-nitrobenzyl ester derivatives of lithocholic, isolithocholic, 3-keto-5β-cholanic, and 5β-cholanic acids were prepared using the reagent o,p-nitrobenzyl-N,N′-diisopropylisourea. Separation was achieved in less than 20 min on a microparticulate silica column using isocratic elution with 2% isopropanol in isooctane as the mobile phase. The p-chlorobenzoyl esters of methylated lithocholic and isolithocholic acids were also prepared but required purification by thin-layer chromatography before separation by hplc. These derivatives were eluted from a Porasil T column using 5% diisopropyl ether in isooctane as the mobile phase. Lithocholic and isolithocholic acids produced by microbial metabolism of [¹⁴COOH]taurolithocholic acid were separated and identified by preparing p-nitrobenzyl derivatives and monitoring the column effluent for both uv and radioactivity. This technique is a rapid and sensitive method for isolating bile acid metabolites.     

Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of ¹³C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of ¹³C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.     

Liver-specific transgenic expression of cholesteryl ester hydrolase reduces atherosclerosis in Ldlr−/− mice

The liver plays a central role in the final elimination of cholesterol from the body either as bile acids or as free cholesterol (FC), and lipoprotein-derived cholesterol is the major source of total biliary cholesterol. HDL is the major lipoprotein responsible for removal and transport of cholesterol, mainly as cholesteryl esters (CEs), from the peripheral tissues to the liver. While HDL-FC is rapidly secreted into bile, the fate of HDL-CE remains unclear. We have earlier demonstrated the role of human CE hydrolase (CEH, CES1) in hepatic hydrolysis of HDL-CE and increasing bile acid synthesis, a process dependent on scavenger receptor BI expression. In the present study, we examined the hypothesis that by enhancing the elimination of HDL-CE into bile/feces, liver-specific transgenic expression of CEH will be anti-atherogenic. Increased CEH expression in the liver significantly increased the flux of HDL-CE to bile acids. In the LDLR^−/− background, this enhanced elimination of cholesterol led to attenuation of diet-induced atherosclerosis with a consistent increase in fecal sterol secretion primarily as bile acids. Taken together with the observed reduction in atherosclerosis by increasing macrophage CEH-mediated cholesterol efflux, these studies establish CEH as an important regulator in enhancing cholesterol elimination and also as an anti-atherogenic target.     

Evidence for the Existence of a Soybean Resistant Protein That Captures Bile Acid and Stimulates Its Fecal Excretion

Feeding HMF, an insoluble “high-molecular-weight fraction” from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestable protein or peptide that can be called a “resistant protein” in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestable fraction of HMF.     

Separation of bile acids as their phenacyl esters by high-pressure liquid chromatography

Bile acids have been separated by high-pressure liquid chromatography. The free acids were derivatized to their phenacyl esters by treatment with triethylamine and α-bromoacetophenone. The stationary phase was a C₁₈, Partisil ODS column. A dual-solvent, stepwise gradient system was used for the mobile phase. The method is applied to a human bile sample and shows excellent resolution of the dihydroxy bile acid phenacyl esters. Detection limits for pure derivatized bile acids are 10–20 pmol (5–10 ng), except for the cholic acid derivative, which has a detection limit of 265 pmol.     

An improved method for the isolation,quantitation, and identification of bile acids in rat feces

An improved method for the isolation and quantitation of bile acids from rat feces was developed. This method employs an initial Soxhlet extraction of the solid fecal material, esterification of the bile acid fraction with dry methanol/HCl and quantitation using a combination of tlc and glc techniques. In addition, identification of the individual components of the fecal bile acid fraction is accomplished by tlc and glc-ms. This method has proven useful for the quantitation and identification of the fecal bile acids during sterol metabolism measurements.     

Rapid quantitation of free fatty acids in human plasma by high-performance liquid chromatography

We report a rapid and sensitive method for separation and quantitation of free fatty acids (FFAs) in human plasma using high-performance liquid chromatography (HPLC). Two established techniques of lipid extraction were investigated and modified to achieve maximal FFA recovery in a reasonably short time period. A modified Dole extraction method exhibited greater recovery (90%) and short processing times (30 min) compared to the method of Miles et al. Reversed-phase HPLC using UV detection was used for plasma FFA separation and quantitation. Two phenacyl ester derivatives, phenacyl bromide and p-bromophenacyl bromide, were investigated in order to achieve optimal separation of individual plasma FFAs (saturated and unsaturated) with desirable detection limits. Different chromatographic parameters including column temperature, column type and elution profiles (isocratic and gradient) were tested to achieve optimal separation and recovery of fatty acids. Phenacyl bromide esters of plasma fatty acids were best resolved using an octadecylsilyl column with endcapped silanol groups. An isocratic elution method using acetonitrile–water (83:17) at 2 ml/min with UV detection at 242 nm and a column temperature of 45°C was found to optimally resolve the six major free fatty acids present in human plasma (myristic [14:0], palmitic [16:0], palmitoleic [16:1], stearic [18:0], oleic [18:1] and linoleic [18:2]), with a run time of less than 35 min and detection limits in the nmol range. The entire process including plasma extraction, pre-column derivatization, and HPLC quantitation can be completed in 90 min with plasma samples as small as 50 μl. Over a wide physiological range, plasma FFA concentrations determined using our HPLC method agree closely with measurements using established TLC–GC methods (r²≥0.95). In addition, by measuring [¹⁴C] or [³H] radioactivity in eluent fractions following HPLC separation of plasma FFA, this method can also quantitate rates of FFA turnover in vivo in human metabolic studies employing isotopic tracers of one or more fatty acids.     

Influence of high-fat diet on host animal health via bile acid metabolism and benefits of oral-fed Streptococcus thermophilus MN-ZLW-002

In this study, C57BL/6J male mice were fed normal chow (NC; control) or a high-fat diet (HFD) for 12 weeks, and HFD mice were supplemented with oral administration of Streptococcus thermophilus MN-ZLW-002 (HFD + MN002); n=20/group. Body weight, visceral fat, blood glucose, blood lipids and liver lipid deposition increased in the HFD group, and the composition of gut microbiota, cecum short-chain fatty acids and fecal bile acids (BAs) also changed. Oral-fed MN-002 increased the relative abundances of Ruminococcaceae, Lachnospiraceae and Streptococcaceae and improved blood glucose, liver cholesterol deposition, and serum IL-10, CCL-3 and the fecal BAs composition. In conclusion, the high-fat diet changed the composition of bile acids by shaping the gut microbiota into an obese type, leading to metabolic disturbances. Streptococcus thermophilus MN-ZLW-002 regulated gut microbiota by adjusting the composition of bile acids and improved the perturbation caused by high-fat diets. However, the effect of MN002 observed in animal experiments needs to be verified by long-term clinical trials.     

General methods for the analysis of metabolic profiles of bile acids and related compounds in feces

A general method is described for the detailed qualitative and quantitative analysis of bile acids and related compounds from feces. The technique utilizes a novel combination of liquid-gel and liquid-solid extraction, lipophilic ion exchange chromatography, and capillary column gas-liquid chromatography coupled to mass spectrometry, which permits the detailed composition of bile acids in feces in terms of both the individual bile acids present and their mode of conjugation in the original fecal sample. The extraction, purification, and isolation procedures have been evaluated using fecal samples containing endogenous radioactive bile acid metabolites and from the addition of radiolabeled standards to fecal homogenates. The applicability of the general procedure is illustrated with examples from the analysis of bile acids and sterols in the feces collected from normal healthy subjects, patients with chronic diarrhea, and an adult female Sprague-Dawley rat. The flexibility of the method, and the general problems encountered in the extraction, purification, and isolation of bile acids and related classes of compounds from feces for subsequent analysis of gas-liquid chromatography are discussed in detail.     

Size exclusion chromatography for extraction of serum bile acids

A major problem in the measurement of serum bile acids is their quantitative extraction from the high molecular protein matrix. In our hands, the standard techniques of adsorption and reversed-phase chromatography have yielded incomplete recovery for different bile acids (33-93%) and poor reproducibility. In contrast, with the novel extraction procedure of size exclusion chromatography, recovery was nearly quantitative (75-104%) and reproducibility was satisfactory. The described method allowed for a reliable determination of serum bile acids in healthy subjects and patients with liver cirrhosis. We conclude that size exclusion chromatography for serum bile acid extraction is more reliable than alternative techniques, because the separation by size is independent of solubility, charge, and polarity.     

Feeding of the water extract from Ganoderma lingzhi to rats modulates secondary bile acids,intestinal microflora,mucins, and propionate important to colon cancer

Consumption of reishi mushroom has been reported to prevent colon carcinogenesis in rodents, although the underlying mechanisms remain unclear. To investigate this effect, rats were fed a high-fat diet supplemented with 5% water extract from either the reishi mushroom (Ganoderma lingzhi) (WGL) or the auto-digested reishi G. lingzhi (AWGL) for three weeks. Both extracts markedly reduced fecal secondary bile acids, such as lithocholic acid and deoxycholic acid (colon carcinogens). These extracts reduced the numbers of Clostridium coccoides and Clostridium leptum (secondary bile acids-producing bacteria) in a per g of cecal digesta. Fecal mucins and cecal propionate were significantly elevated by both extracts, and fecal IgA was significantly elevated by WGL, but not by AWGL. These results suggest that the reishi extracts have an impact on colon luminal health by modulating secondary bile acids, microflora, mucins, and propionate that related to colon cancer.     

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.     

Distinct signatures of host–microbial meta-metabolome and gut microbiome in two C57BL/6 strains under high-fat diet

A combinatory approach using metabolomics and gut microbiome analysis techniques was performed to unravel the nature and specificity of metabolic profiles related to gut ecology in obesity. This study focused on gut and liver metabolomics of two different mouse strains, the C57BL/6J (C57J) and the C57BL/6N (C57N) fed with high-fat diet (HFD) for 3 weeks, causing diet-induced obesity in C57N, but not in C57J mice. Furthermore, a 16S-ribosomal RNA comparative sequence analysis using 454 pyrosequencing detected significant differences between the microbiome of the two strains on phylum level for Firmicutes, Deferribacteres and Proteobacteria that propose an essential role of the microbiome in obesity susceptibility. Gut microbial and liver metabolomics were followed by a combinatory approach using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and ultra performance liquid chromatography time of tlight MS/MS with subsequent multivariate statistical analysis, revealing distinctive host and microbial metabolome patterns between the C57J and the C57N strain. Many taurine-conjugated bile acids (TBAs) were significantly elevated in the cecum and decreased in liver samples from the C57J phenotype likely displaying different energy utilization behavior by the bacterial community and the host. Furthermore, several metabolite groups could specifically be associated with the C57N phenotype involving fatty acids, eicosanoids and urobilinoids. The mass differences based metabolite network approach enabled to extend the range of known metabolites to important bile acids (BAs) and novel taurine conjugates specific for both strains. In summary, our study showed clear alterations of the metabolome in the gastrointestinal tract and liver within a HFD-induced obesity mouse model in relation to the host–microbial nutritional adaptation.     

Bile acid and sterol metabolism with combined HMG-CoA reductase and PCSK9 suppression

Proprotein convertase subtilisin-kexin-9 (PCSK9) inhibition markedly augments the LDL lowering action of statins. The combination is being evaluated for long-term effects on atherosclerotic disease outcomes. However, effects of combined treatment on hepatic cholesterol and bile acid metabolism have not yet been reported. To study this, PCSK9-Y119X mutant (knockout) and wild-type mice were treated with or without atorvastatin for 12 weeks. Atorvastatin progressively lowered plasma LDL in each group, but no differences in liver cholesterol, cholesterol ester, or total bile acid concentrations, or in plasma total bile acid levels were seen. In contrast, atorvastatin increased fecal total bile acids (∼2-fold, P < 0.01) and cholesterol concentrations (∼3-fold, P < 0.01) versus controls for both PCSK9-Y119X and wild-type mice. All 14 individual bile acids resolved by LC-MS, including primary, secondary, and conjugated species, reflected similar increases. Expression of key liver bile acid synthesis genes CYP7A1 and CYP8B1 were ∼2.5-fold higher with atorvastatin in both strains, but mRNA for liver bile acid export and reuptake transporters and conjugating enzymes were not unaffected. The data suggest that hepatocyte cholesterol and bile acid homeostasis is maintained with combined PCSK9 and HMG-CoA reductase inhibition through efficient liver enzymatic conversion of LDL-derived cholesterol into bile acids and excretion of both, with undisturbed enterohepatic recycling.     

Differential impact of hepatic deficiency and total body inhibition of MTP on cholesterol metabolism and RCT in mice

Because apoB-containing lipoproteins are pro-atherogenic and their secretion by liver and intestine largely depends on microsomal triglyceride transfer protein (MTP) activity, MTP inhibition strategies are actively pursued. How decreasing the secretion of apoB-containing lipoproteins affects intracellular rerouting of cholesterol is unclear. Therefore, the aim of the present study was to determine the effects of reducing either systemic or liver-specific MTP activity on cholesterol metabolism and reverse cholesterol transport (RCT) using a pharmacological MTP inhibitor or a genetic model, respectively. Plasma total cholesterol and triglyceride levels were decreased in both MTP inhibitor-treated and liver-specific MTP knockout (L-Mttp^−/−) mice (each P < 0.001). With both inhibition approaches, hepatic cholesterol as well as triglyceride content was consistently increased (each P < 0.001), while biliary cholesterol and bile acid secretion remained unchanged. A small but significant decrease in fecal bile acid excretion was observed in inhibitor-treated mice (P < 0.05), whereas fecal neutral sterol excretion was substantially increased by 75% (P < 0.001), conceivably due to decreased intestinal absorption. In contrast, in L-Mttp^−/− mice both fecal neutral sterol and bile acid excretion remained unchanged. However, while total RCT increased in inhibitor-treated mice (P < 0.01), it surprisingly decreased in L-Mttp^−/− mice (P < 0.05). These data demonstrate that: i) pharmacological MTP inhibition increases RCT, an effect that might provide additional clinical benefit of MTP inhibitors; and ii) decreasing hepatic MTP decreases RCT, pointing toward a potential contribution of hepatocyte-derived VLDLs to RCT.     

Modulation of bile acid profile by gut microbiota in chronic hepatitis B

Chronic hepatitis B (CHB) is a global epidemic disease that may progress to fibrosis, cirrhosis and hepatocellular carcinoma. The role of the liver‐bile acid‐microbiota axis in CHB remains unclear. The aims of this study are to elucidate the alteration of the gut microbiota and its functions in bile acid homeostasis in CHB patients with different degrees of fibrosis. In the present study, we evaluated serum and faecal bile acid profiles in healthy controls and CHB patients with biopsy‐proven diagnosis: patients had stage 0‐1 fibrosis were classified as mild CHB and patients had stage 2‐4 fibrosis were classified as moderate/advanced CHB. The levels of serum total bile acids (BAs) and primary BAs were increased in CHB patients with moderate/advanced fibrosis, whereas faecal total and secondary BAs levels were significantly lower. Analyses of gut microbiota exhibited a trend of decreased abundance in bacteria genera responsible for BA metabolism in CHB patients with moderate/advanced fibrosis. CHB is associated with altered bile acid pool which is linked with the dysregulated gut microbiota. The higher level of FGF‐19 may act in a negative feedback loop for maintaining the bile acid homeostasis.     

Chemical synthesis of bile acid acyl-adenylates and formation by a rat liver microsomal fraction

In mammals, unconjugated bile acids formed in the intestine by bacterial deconjugation are reconjugated (N-acylamidated) with taurine or glycine during hepatocyte transport. Activation of the carboxyl group of bile acids to form acyl-adenylates is a likely key intermediate step in bile acid N-acylamidation. To gain more insight into the process of bile acid adenylate formation, we first synthesized the adenylates of five common, natural bile acids (cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and lithocholic acid), and confirmed their structure by proton NMR. We then investigated adenylate formation by subcellular fractions of rat liver (microsomes, mitochondria, cytosol) using a newly developed LC method for quantifying adenylate formation. The highest activity was observed in the microsomal fraction. The reaction required Mg²⁺ and its optimum pH was about pH 7.0. In term of maximum velocity (V_max) and the Michaelis constant (K_m), the catalytic efficiency of the enzyme under the conditions used was highest with cholic acid of the bile acids tested. The formation of cholyl-adenylate was strongly inhibited by lithocholic and deoxycholic acid, as well as by palmitic acid; ibuprofen and valproic acid were weak inhibitors. In cholestatic disease, such adenylate formation might lead to subsequent bile acid conjugation with glutathione or proteins.     

Intestinal CYP3A4 protects against lithocholic acid-induced hepatotoxicity in intestine-specific VDR-deficient mice

Vitamin D receptor (VDR) mediates vitamin D signaling involved in bone metabolism, cellular growth and differentiation, cardiovascular function, and bile acid regulation. Mice with an intestine-specific disruption of VDR (Vdr^ΔIEpC) have abnormal body size, colon structure, and imbalance of bile acid metabolism. Lithocholic acid (LCA), a secondary bile acid that activates VDR, is among the most toxic of the bile acids that when overaccumulated in the liver causes hepatotoxicity. Because cytochrome P450 3A4 (CYP3A4) is a target gene of VDR-involved bile acid metabolism, the role of CYP3A4 in VDR biology and bile acid metabolism was investigated. The CYP3A4 gene was inserted into Vdr^ΔIEpC mice to produce the Vdr^ΔIEpC/3A4 line. LCA was administered to control, transgenic-CYP3A4, Vdr^ΔIEpC, and Vdr^ΔIEpC/3A4 mice, and hepatic toxicity and bile acid levels in the liver, intestine, bile, and urine were measured. VDR deficiency in the intestine of the Vdr^ΔIEpC mice exacerbates LCA-induced hepatotoxicity manifested by increased necrosis and inflammation, due in part to over-accumulation of hepatic bile acids including taurocholic acid and taurodeoxycholic acid. Intestinal expression of CYP3A4 in the Vdr^ΔIEpC/3A4 mouse line reduces LCA-induced hepatotoxicity through elevation of LCA metabolism and detoxification, and suppression of bile acid transporter expression in the small intestine. This study reveals that intestinal CYP3A4 protects against LCA hepatotoxicity.