Determination of individual conjugated bile acids in human bile

A method has been developed and validated for the determination of the six major conjugated bile acids, cholesterol, and total phospholipids in bile of human subjects previously injected with 4-(14)C-cholesterol. The procedure is designed for use with 5-10 ml of duodenal or T-tube bile and eliminates difficulties associated with existing methods for bile acid determination, in particular the requirement for preliminary saponification under pressure or the use of paper chromatography. Saponification under pressure is employed only in steps where partial destruction of the steroid moiety of conjugated bile acids is not a crucial matter. A preliminary Folch extraction and washing step separated free cholesterol and phospholipids (bottom layer) from the six major conjugated bile acids (top layer). The conjugated bile acids were then fractionated cleanly by thin-layer chromatography to give four groups, the (14)C content of each of which was determined. A second aliquot of the top layer was used to determine (after deconjugation) the radioactivity ratio of deoxycholic acid to chenodeoxycholic acid for the two unresolved groups (dihydroxycholanoic acid conjugates with glycine and taurine, respectively). A third aliquot was used for determination of specific activities of the methyl esters of cholic, chenodeoxycholic, and deoxycholic acids derived from the total bile salts. Appropriate calculations yielded the concentration in bile of all six major bile acid conjugates.     

The determination and excretion of individual human fecal bile acids

A fluorometric method, using resazurin, for the analysis of individual fecal bile acids separated by thin layer chromatography of crude fecal extracts is described. The method is precise and accurate. The assay was used to investigate the constancy of excretion of individual fecal bile acids in small random stool samples collected over three weeks in six humans. Relatively small but significant variations were found in each case, most were random but one was progressive.     

Rapid analysis of human fecal bile acids

A rapid, accurate, precise method for determining human fecal bile acids is reported. Feces are homogenized and then briefly extracted with boiling absolute ethanol. A portion of the extract is evaporated to dryness and the residue heated with mild alkali to hydrolyze bile acid 3α-hydroxyl esters. Aliquots of hydrolyzed crude extract are treated with resazurin reagent which effects a series of enzyme catalyzed reactions in which bile acid free 3α-hydroxyls are first oxidized to 3-oxo-groups in a reaction catalyzed by 3α-hydroxysteroid dehydrogenase. Resulting protons are transferred to β-nicotinamide adenine dinucleotide, yielding reduced β-nicotinamide adenine dinucleotide (β-NADH). β-NADH then reduces nonfluorescent resazurin to fluorescent resorufin in a reaction catalyzed by diaphorase. Developed fluorescence, which is proportional to the extract aliquots bile acid content, is excited at 565 nm and read at 580 nm, wavelengths which lie in a spectral region in which there is minimal fecal pigment absorption. 3-Oxo-bile acids and bile acid 3α-sulfates are extracted in the procedure but reduction and/or solvolysis is necessary before quantification.     

Identification of unconjugated bile acids in human bile

Unconjugated bile acids in the bile of healthy and diseased humans were determined qualitatively and quantitatively by means of gas-liquid chromatography and gas-liquid chromatography-mass spectrometry, after their isolation by ion-exchange chromatography. In a healthy person and three patients with cholelithiasis, unconjugated bile acids comprised 0.1-0.4% of total biliary bile acids. The bile acid composition of the unconjugated fraction was quite different from that of the glycine- or taurine-conjugate fraction, in that it contained a relatively large proportion of unusual bile acids including C23 and C27 bile acids. In two patients with cerebrotendinous xanthomatosis, C22 and C23 bile acids were the major constituents of the biliary unconjugated bile acids, and comprised about 0.8% of total bile acids; no detectable amounts of C27 bile acids were found in their bile. The analysis of biliary unconjugated bile acids may be useful for the diagnosis of metabolic diseases concerning bile acids, particularly the accumulation or disappearance of unusual bile acids.     

A proteomic analysis of human bile

We have carried out a comprehensive characterization of human bile to define the bile proteome. Our approach involved fractionation of bile by one-dimensional gel electrophoresis and lectin affinity chromatography followed by liquid chromatography tandem mass spectrometry. Overall, we identified 87 unique proteins, including several novel proteins as well as known proteins whose functions are unknown. A large majority of the identified proteins have not been previously described in bile. Using lectin affinity chromatography and enzymatically labeling of asparagine residues carrying glycan moieties by (18)O, we have identified a total of 33 glycosylation sites. The strategy described in this study should be generally applicable for a detailed proteomic analysis of most body fluids. In combination with "tagging" approaches for differential proteomics, our method could be used for identification of cancer biomarkers from any body fluid.     

A non-chromatographic radioimmunoassay of 18-hydroxy-11-deoxycorticosterone in human plasma

A method is described for a non-chromatographic assay of 18 hydroxy-11-deoxycorticosterone (18-OH-DOC) using a sensitive and specific antiserum. This direct measurement is assessed in terms of accuracy and precision. The mean 8a.m. plasma 18-OH-DOC levels in the supine position was 10.1 ± 6.5 ng per 100ml in 20 normal subjects and 9.4 ± 4.2 ng per 100ml after two hours of movement. These values are correlated with those obtained in aldosterone. The ACTH-dependency of 18-OH-DOC is demonstrated by diurnal variation and treatment with dexamethasone.     

A specific, non-chromatographic radioimmunoassay for human plasma cortisol.

A radioimmunoassay technique has been developed for the measurement of cortisol in a single methylene chloride extract of human plasma without chromatography. The antiserum, obtained by immunizing rabbits with cortisol-3-carboxymethyl-oxime conjugated to bovine serum albumin, had a high affinity (KA = 1.8 X 10(9) 1/mole) and capacity (2.3 X 10(-6) moles/L undiluted serum) for cortisol. The minimum detectable amount determined at the lower 95% confidence limit of the buffer control tubes was 8.3 +/- 4.7 pg/tube and a log dose - logit response standard curve was linear between 20 pg and 20 ng/tube. The antiserum was highly specific for cortisol with only corticosterone, cortisone, 11-deoxycortisol and 21-deoxycortisol showing significant cross-reaction (12.4, 6.6, 3.8 and 3.7%, respectively). The cross-reaction for the other tested naturally occurring and synthetic steroids did not exceed 1%. Regression analysis of cortisol concentration estimates obtained on 20 samples before and after Sephadex LH-20 column chromatography gave a coefficient of correlation (r) of 0.995 and a regression coefficient (b) of 1.04. Recovery of cortisol added to plasma samples was quantitative. The intra-assay error was 8.5% and the inter-assay error averaged 5.7%. The method is simple requiring a single solvent extraction of plasma, therefore permitting large numbers of samples to be handled efficiently by a single technician.     

High pressure liquid chromatographic analysis of conjugated bile acids in human bile: simultaneous resolution of sulfated and unsulfated lithocholyl amidates and the common conjugated bile acids

A reversed phase high pressure liquid chromatography (HPLC) system capable of simultaneously separating four lithocholyl species (sulfated and unsulfated forms of lithocholylglycine and lithocholyltaurine) as well as the eight other major conjugated bile acids present in human bile is described. The system uses a C18 octadecylsilane column and isocratic elution with methanol phosphate buffer, pH 5.35. Relative bile acid concentration is determined by absorbance at 200 nm. Retention times relative to chenodeoxycholylglycine are reported for the four lithocholic acid forms, the glycine and taurine amidate of the four major bile acids present in human bile (cholic, chenodeoxycholic, ursodeoxycholic, and deoxycholic), and for their corresponding unconjugated forms. Retention times are also reported for the glycine and taurine amidates as well as the unconjugated form of the C23 norderivatives of these bile acids. Maximal absorbance of bile acid amidates is at 200 nm and is very similar for the (unsulfated) glycine and taurine amidates. Sulfated lithocholyl amidates exhibit molar absorptivities at 200 nm which are 1.4 times greater than that of non-sulfated lithocholyl amidates. Unconjugated bile acid absorbance at 200 nm or 210 nm is 20 to 30 times less than that of corresponding peptide conjugates. The method has been applied to samples of gallbladder bile obtained from 14 healthy subjects to define the pattern of conjugated bile acids present in human bile.     

A rapid and sensitive method for the assay of UDP-glucuronosyltransferase activity toward bile acids

A rapid and sensitive procedure is described for the assay of rat liver microsomal UDP-glucuronosyltransferase activity toward the bile acids chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, and lithocholic acid using the radioactively labeled bile acids as substrates. The unreacted bile acids were separated from the bile acid glucuronides formed as products of the enzymatic reactions by extraction with chloroform, leaving the bile acid glucuronides in the aqueous phases. The bile acid glucuronides were characterized by their mobilities in thin-layer chromatography and identified by their sensitivity to hydrolysis with β-glucuronidase and inhibition of hydrolysis by the specific β-glucuronidase inhibitor d-saccharic acid-1,4-lactone. Enzyme activities were optimal at pH 6.8 and were maximally stimulated about fourfold by the addition of the nonionic detergent Brij 58 at a concentration of 0.3 mg/mg microsomal protein. The kinetic parameters for the various bile acids as substrates were determined.     

A non-chromatographic radioimmunoassay of prugesterone in serum

Convenient methodology based on separation of progesterone from alcoholic neutral steroids by means of a sulfation-procedure has been developed for the radioimmunoassay (RIA) of progesterone in male and female serum. When coordinated with our previously published nonchromatographic procedure for the RIA of estrone and estradiol in serum, all 3 seteroids can be determined in the same specimen. Validation of the procedure was based on: 1. Agreement between results obtained using TLC and sultation to fractionate progesterone (r=0.98; b=0.86), 2. accurate recovery of different quantities of progesterone added to serum, 3. independence of the concentration of progesterone and volume of serum used for assay, 4. low procedural blanks (3.6 ± 1.3 pg), 5. low intraassay (9.7 – 10.3%) and interassay (11.0 – 11.6%) variability and 6. correspondence of observed values for progesterone in male serum (108 ± 20 pg/ml) and in female serum (follicular, 285 ± 149 pg/ml; luteal, 3.46±1.45 ng/ml) with those reported previously by others.     

Determination of intralumenal individual bile acids by HPLC with charged aerosol detection

An isocratic HPLC charged aerosol detector (CAD) method was developed, validated, and applied for the determination of individual bile acids in human gastric and duodenal aspirates. The method requires a low volume of aspirates (50-100 microl) and minimal sample pretreatment. A Hypersil BDS RP-C(18) column (250 x 4.6 mm, 5 microm particle size) was equilibrated with a mobile phase composed of methanol-[ammoniun formate 20 mM, formic acid 0.5%, triethylamine 0.2% (pH 3)] 67:33 v/v. Its flow rate was 1 ml/min. The elution times for taurocholate, glycocholate, taurochenodeoxycholate, ursodeoxycholate, glycochenodeoxycholate, cholate, and glycodeoxycholate were approximately 9.9, 16.2, 18.2, 21.3, 31.6, 34.5, and 38.5 min, respectively. Calibration curves in the mobile phase were constructed in the concentration range of 0.5-500 microM. Limits of detection and quantification were in the range of 0.07-0.60 microM and 0.20-1.80 microM, respectively. This method was applied first, in gastric aspirates collected in the fasted state, in which bile acid presence is minimal and, second, in duodenal aspirates collected in the fed state, in which a large number of potentially interfering compounds exists. Intra-day relative standard deviation in fasted gastric aspirates and in fed duodenal aspirates was less than 2.2% and 6.0%, respectively.     

A novel binding site for bile acids on human serum albumin

Binding sites of bile acids on human serum albumin were studied using various probes: dansylsarcosine (site I probe), 7-anilinocoumarin-4-acetic acid (ACAA, site II probe), 5-dimethylaminonaphthelene-1-sulfonamide (DNSA, site III probe), cis-parinaric acid (probe for fatty acid binding site) and bilirubin. Bile acids competitively inhibited the binding of dansylsarcosine to human serum album whereas bile acids enhanced the binding of ACAA, DNSA, cis-parinaric acid and bilirubin. Considering the concentrations of bile acids required to inhibit the binding of dansylsarcosine to human serum albumin, the secondary binding site of bile acids may correspond to site I. Dissociation constants (K_d) of the primary binding sites of lithocholic and chenodeoxycholic acid to human serum albumin were approximately 0.2 and 4 μM, respectively, which was measured by equilibrium dialysis at 37° C. All the bile acids and their sulfates and glucuronides inhibited the binding of chenodeoxycholic acid to human serum albumin. Lithocholic and chenodeoxycholic acid and their sulfates and glucuronides exhibited more inhibition than cholic acid and its conjugates. In conclusion, bile acids may bind to a novel binding site on human serum albumin.     

Gas-liquid chromatographic determination of human fecal bile acids

A method for the determination of total bile acids in human feces that is suitable for routine application is described and discussed. Bile acids are extracted from freeze-dried feces with acetic acid and toluene, in the presence of the internal standard 23-nordeoxycholic acid. After saponification of the extract, bile acids and the internal standard are methylated and converted by mild chromic acid oxidation into their ketonic derivatives. The resultant mixture of a few stable compounds can be separated and measured quantitatively by gas-liquid chromatography on a methylsiloxane polymer. A reference bile acid mixture including the internal standard is also taken through the entire procedure with each series of samples. It has been demonstrated that, in spite of the omission of the usual purification steps, the method is specific for bile acids.     

A step further in the analysis of human bile proteome

Bile was shown to collect proteins known as potential cancer biomarkers. Thorough proteomic analysis of bile is of particular interest to search for new, more sensitive and more specific, biomarkers of cancers affecting the biliary tract and surrounding organs, such as the pancreas and the liver. Therefore, extending the knowledge of the bile proteome is highly relevant, but this has proved technically difficult. In this study, we describe a strategy that circumvents problems related to the biochemical complexity of this sample and the presence of high concentrations of interfering substances. Bile collected from a patient suffering from a biliary stenosis caused by a pancreatic adenocarcinoma was fractionated by a differential centrifugation scheme, involving a stepwise increase in centrifugation speeds. Pellets and the final supernatant were further fractionated by polyacrylamide gel electrophoresis and proteins were in-gel digested prior to LC-MS/MS analysis. This approach allowed the identification of 445 unique proteins with at least two peptides (812 proteins if single-hit proteins were included), which represents a 3-fold increase in the knowledge of bile proteome. The subsequent literature comparison revealed that numerous biliary proteins identified in this sample were related to pancreas cancer. Immunoblot analysis of some known tumor markers revealed that they were preferentially associated with the soluble fraction rather than with pellets containing cellular components.     

Presence of monohydroxy bile acids in the urinary precipitates: a pitfall in the analysis of urinary bile acids

The importance of the method of handling the urinary precipitates frequently present in urine samples, especially after freezing-thawing, for bile acid analysis is emphasized because of the presence of a considerable proportion of monohydroxy bile acids such as lithocholic and 3 beta-hydroxy-5-cholenoic acids. Filtration of the urinary precipitates may lead to the underestimation of these important bile acid species.