Separation by thin-layer chromatography and structure elucidation of bilirubin conjugates isolated from dog bile.

1. A system for separation of bile pigments by t.l.c. and for their structure elucidation is presented. Separated bile pigments are characterized by t.l.c. of derived dipyrrolic azopigments. 2. At the tetrapyrrolic stage hydrolysis in strongly alkaline medium followed by t.l.c. demonstrates the presence of bilirubin-IIIalpha, -IXalpha and -XIIIalpha and allows assessment of their relative amounts. 3. Most structural information is derived from analysis of dipyrrolic azopigments. Such derivatives, obtained by treatment of separated bile pigments with diazotized ethyl anthranilate, were separated and purified by t.l.c. Micro methods showed (a) the nature of the dipyrrolic aglycone, (b) the nature of the bonds connecting aglycone to a conjugating group, (c) the ratio of vinyl/isovinyl isomers present in the aglycone and, (d) the nature of the conjugating groups (by suitable derivative formation and t.l.c. with reference to known compounds). 4. In bile of normal dogs at least 20 tetrapyrrolic, diazo-positive bile pigments could be recognized. Except for two pigments the tetrapyrrolic nucleus corresponded predominantly to bilirubin-IXalpha. All conjugated pigments had their conjugating groups connected in ester linkage to the tetrapyrrolic aglycone, Apart from bilirubin-IXalpha, monoconjugates and homogeneous and mixed diconjugates of bilirubin were demonstrated; conjugating groups of major importance were xylose, glucose and glucuronic acid. 5. Bilirubin isomer determination on native bile and isolated bile pigments, and dipyrrole-exchange assays with [14C8]bilirubin indicated (a) that the conjugates pre-exist in bile, and (b) that no significant dipyrrole exchange occurs during isolation of the pigments.     

Separation of bile acids of rat bile by thin-layer chromatography

The common bile acids of rat bile (chenodeoxycholic, hyodeoxycholic, cholic, alpha-muricholic, and beta-muricholic acids) are completely separated by a new thin-layer chromatographic system.     

Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography

Class separation of methylated free bile acids from bile acids conjugated with taurine and methylglycine was accomplished using a solvent system of 2,2,4-trimethylpentane-absolute ethanol 10:1 (v/v). By developing a silica thin-layer plate two times with solvent in a Brinkmann sandwich tank, the difficult resolution between methyl cholate and methyl glycolithocholate was achieved. Evidence is presented that this separation system may be useful as a preparative step in the analysis of bile acids by gas-liquid chromatography or high pressure liquid chromatography.--Bolt, M. J. G. Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography.     

Bilirubin conjugates in bile of man, rat and dog. Semi-quantitative analysis of bile composition by thin-layer chromatography.

1. Conjugated bile pigments, separated in two fractions by semi-quantitative t.l.c. performed on silicic acid with phenol/water as the developing solvent, were treated with diazotized ethyl anthranilate. Resulting dipyrrylazo derivatives were analysed by quantitative t.l.c. 2. The tentative structure elucidation of tetrapyrrolic bilirubin conjugates and semi-quantitative evaluation of rat bile, post-obstructive human bile and dog bile composition is presented. 3. Homogeneous and mixed hexuronic acid diesters of bilirubin containing glucuronic acid constitute 51% of the total conjugates in normal rat bile, 45% of those in human post-obstructive bile and 38% of those in obstructed rat biles. 4. Monoconjugated bilirubin amounts to 33% of total conjugated bile pigments in normal rat bile, and 17 and 14% in post-obstructive hepatic human bile and gall-bladder bile of dog respectively. After loading with unconjugated bilirubin a greater amount of monoconjugates (56%) occur in the rat bile, whereas bilirubin diglucuronide excretion is decreased (34%). 5. In gall-bladder bile of normal dog, 40% of glucose-containing diconjugates, 32% of homogeneous and/or mixed hexuronic acid (mainly glucuronic acid) diesters of bilirubin and 14% of xylose-containing diconjugates are estimated. 6. Increased amounts of bilirubin conjugates, including some with unidentified uronic acid groups, were observed in cholestatic rat biles and quantities of conjugates with glucuronic acid were decreased.     

Assay for taurine conjugates of bile acids in serum by reversed-phase high-performance liquid chromatography

The purpose of this study was to develop a new high-performance liquid chromatographic (HPLC) procedure for quantifying taurine conjugates of bile acids in serum. The technique involved three basic steps. The first removed free amino acids via solid-phase extraction of the serum. The second step involved the reaction of the extracted serum with the enzyme choloylycine hydrolase, which liberated the taurine from the conjugated bile acids. The third step was the reversed-phase HPLC separation of o-phthalicdicarboxaldehhyde derivatives of taurine. The assay provides a simple technique for determination of the total amount of taurine-conjugated bile acids in serum.     

Separation of glycoprotein-derived oligosaccharides by thin-layer chromatography.

A thin-layer chromatography (tlc) system has been developed for the separation of glycoprotein-derivedoligosaccharides. The method involves chromatography on silica gel using n-propanol/acetic acid/water (3:3:2 v/v) as the solvent. This tlc method was used to separate pathological oligosaccharides isolated from individuals with G_M1 gangliosidosis and with neuraminidase deficiency. The results indicate the potential usefulness of the system in the analysis of complex carbohydrates.     

Separation of bilirubin species in serum and bile by high-performance reversed-phase liquid chromatography

A high-performance, reversed-phase liquid chromatographic (HPLC) procedure has been developed for the separation of at least three major bilirubin fractions in bile and four fractions in human serum. This procedure was unlike most others, in that serum was not totally deproteinized prior to injection onto the HPLC column; instead, serum was treated with an excess of sodium sulfate solution to precipitate primarily proteins larger than albumin. Injection of the filtered and diluted supernatant onto a reversed-phase column then resulted in the separation of the bilirubin species in a 24-min gradient elution run. Both the initial aqueous acidic mobile phase and the final isopropyl alcohol-based mobile phase contained 5% methoxyethanol (v/v) to facilitate elution of albumin still present in the treated sample. Bilirubin species eluting from the column were detected by absorbance at 450 nm.Results of a number of chromatographic separations of pathological sera indicated a wide variation in the relative proportions of the four bilirubin fractions observed. A correlation of the sum of the areas of the bilirubin peaks observed by HPLC was found with the total bilirubin value obtained by a standard reference procedure.     

Improved separation of sterols by reversed-phase thin-layer chromatography

Some closely related sterols have been separated with better resolution in a shorter period of time than has previously been reported. Separations were effected on the basis of carbon number and the number and location of double bonds through the use of paraffin-impregnated kieselguhr chromatoplates in the system (paraffin oil)/(acetone-water 4:1).     

Separation and purification of xylose oligomers using centrifugal partition chromatography

Xylose oligomers, which have a prebiotic effect, have been used as additives to human and animal food. These oligomers are also the primary intermediate in hemicellulose degradation during the pretreatment of biomass. Centrifugal partition chromatography (CPC) was used in this study to separate and purify xylan-derived oligomers from birchwood xylan. The xylan was partially hydrolyzed to achieve varying degrees of polymerization at 130°C using 0.98% aqueous sulfuric acid for 20 min with a 2.5% solid loading. The CPC solvent system consisting of dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and water in a 1:6:3 volumetric ratio was used because of its ability to dissolve xylose oligomers of different degrees of polymerization. The CPC was operated in the ascending mode with the water- and DMSO-rich bottom phase acting as the stationary phase, while the THF-rich top phase was the eluent. This paper delineates a method for the production and purification of xylose monomer and xylose oligomers (up to xylopentaose) using CPC. The amount and purity of compounds collected from the CPC fractionation based on 1 g of birchwood xylan were 25.26 mg of xylose at 91.86% purity, 10.71 mg of xylobiose at 85.07% purity, 4.15 mg of xylotriose at 54.71% purity, 5.03 mg of xylotetraose at 38.33% purity and 3.31 mg of xylopentaose at 30.43% purity.     

A fast and simple radiometric assay for adenosine deaminase using reversed-phase thin-layer chromatography

A new quantitative radiometric assay for adenosine deaminase is described. The reaction conditions are similar to those used in other radioassays and are shown to result in an activity which increases linearly with time and with enzyme concentration. An original feature of the technique resides in the use of reversed-phase thin-layer chromatography to separate adenosine from inosine. The separation is complete, fast, and reproducible. Both compounds can be recovered almost quantitatively from the plates. The assay is very simple and allows the determination of up to 36 samples in 3 h.     

Separation of adreno-ovarian steroids by thin-layer chromatography

The major adreno-ovarian steroid hormones (progesterone, estrone, 17α-estradiol, 17β-estradiol, estriol, corticosterone, cortisone, and cortisol) have been separated simultaneously on a single TLC plate without recourse to transfer chromatography. The plate was developed successively twice in benzene/ethanol (95:5, v/v) solvent system. It was then sprayed with rhodamine 6G and a line was drawn isolating the already separated least polar and medium-polar steroids (progesterone, estrone, 17α-estradiol, and 17β-estradiol) with the help of ultraviolet light. Then 5 ml methanol per 100 ml solvent in the tank was added and the plate again developed 2–3 times up to the line drawn, when polar steroids (corticosterone, cortisone, cortisol, and estriol) separated out.     

Separation of sterols by vapor-programmed thin-layer chromatography

By vapor-programmed thin-layer chromatography on silica gel, it was possible to separate cholestanol from cholesterol and stigmastanol from beta-sitosterol. The method was applied for the analysis of beta-sitosterol-3-(14)C.     

Revisited mycolic acid pattern of Mycobacterium confluentis using thin-layer chromatography

The profile of mycolic acids from Mycobacterium confluentis has not been adequately published. However, the definition of the composition of mycolic acids is a critical element for describing new mycobacterial species. Thus, an erroneously published profile can lead to confusing citations. The aim of this article is to make the protocols clear, by using thin layer chromatography as a tool, for defining the discrete pattern of mycolic acids of any newly reported mycobacterial species. By using this method, and corroborated using nuclear magnetic resonance analysis, we demonstrated that M. confluentis contains α-mycolates (type I) and epoxymycolates (type V mycolic acids).     

The hydrolysis of bile acid conjugates

Studies were made of a) the relationship of bile acid structure and analytical recoveries (measured by 3-hydroxysteroid oxidoreductase) following vigorous alkaline hydrolysis of bile acid conjugates and b) the relationship of structure and hydrolysis time of taurine- and glycine bile acid conjugates in a reaction catalyzed by glycocholic acid hydrolase. Alkaline hydrolysis resulted in good recoveries of hydroxy and 7 and 12- oxo-bile acids but poor recoveries of 3-oxo-bile acids. Borohydride reduction of the 3-oxo-acids prevented these losses. Complete enzymatic hydrolysis of glycine conjugated bile acids was about five times more rapid than that of taurine conjugates. Hydrolysis of conjugates containing oxo groups was slow. Borohydride reduction of oxoacids corrected this and did not inhibit enzymatic hydrolysis. It was concluded that both vigorous alkaline and enzymatic hydrolysis are satisfactory in bile acid assays if borohydride reduction is instituted before the hydrolytic step. However, due to the presence of possible enzyme inhibitors and solubility difficulties, strong alkaline hydrolysis is preferable to enzymatic hydrolysis in fecal bile acid determinations at this time.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.