Glycine-Conjugated BAs

With pKas of 3.8 to 4.8, these are the most abundant conjugated bile acids, (representing 70% of bile). It should be noted that conjugation restricts their entry into the epithelial cells, ensuring that they remain within the intestinal lumen and do not leak into the intra-cellular spaces to damage other organs. However, at pH values approaching their pKa values, they become un-ionised and can cross intestinal membranes to a certain extent. 

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Free and glycine-conjugated BAs are only slightly soluble in acid solutions. As the pH is increased, the solubility will increase. This is a very important characteristic since it describes the solubility characteristics of the major BAs, and, it also explains their potential to enter the epithelium at physiological pH ranges. 

Standard solutions of free BAs and of glycine-conjugated Bas are employed for the calibration. In the cited work the following concentration ranges were used 0.47-... 

The chromatogram of free BA standard mixture is reported in Fig. 5.4.7. The Br-AMN degradation products are eluted at lower retention times than derivatised BA, close to the solvent front, so they do not impair BA separation. Free BA fraction also encloses taurine conjugates, previously enzymatically hydrolysed. The separation of glycine conjugated BA is illustrated in Fig. 5.4.8. In both chromatograms, the peaks of BA naphthacyl esters are fully resolved and separated from the reagent peaks. 

This CE method provides an efficient approach for rapid and effective separation of serum conjugated BAs with an analysis time of 8 min. It is important to mention that each micellar solution plays an important role in the analysis use of 20 mM SDS is to modify the electro-osmotic flow, whereas solubility of glycine-conjugated BAs and the peak shape of all BAs are maintained with 20% acetonitrile and the neutral pH of the phosphate buffer. The optimum condition for baseline separation is achieved with the addition of 8 mM CD. 

Identification of taurine- and glycine-conjugated BAs is based on the integral of the corresponding peak of total conjugated BAs at 7.8-8.1 ppm (signal of amide). Total... 

Stock solutions of free, and glycine- and taurine-conjugated BA in methanol. 

The possibility of defining serum BA composition was also assessed in the reported study. Analysis of the free fraction before and after treatment with ampicillin of a patient with PBC was performed (Fig. 5.4.9). The antibiotics are used in the therapy of cholestatic syndrome, but some studies have been carried out to define their use. This kind of therapy inhibits the production of secondary BAs (DCA and LCA) and the deconjugation of taurine and glycine conjugates. Consequently, an... 

Table 5.4.14 Mobile phase gradient composition used for simultaneous separation of free, and glycine- and taurine-conjugated BA within a single run (adapted from [33])...

For quantitative estimation, a sealed reusable capillary tube, with a known quantity of sodium salt of trimethylsilyl propionic acid (TSP) dissolved in 35 pi of D20, is inserted into the NMR tube while obtaining NMR spectra. The internal standard TSP is used as a chemical shift reference as well as a quantitative standard for the estimation of metabolites, and D20 is used as the field-frequency-lock . Spectra are acquired at room temperature. Typical spectra acquired at room temperature of human bile and standard glycine- and taurine-conjugated BAs are shown in Fig. 5.4.16. 

BA, bile acids G, glycine conjugate T, taurine conjugate C, cholic acid CD, chenodeoxycholic acid DC, deoxycholic acid —, decreased increased =, unchanged,... 

Human serum sample (0.5 ml), diluted with 2.5 ml of 0.1 N NaOH is incubated at 65°C for 15 min. The free, and glycine- and taurine-conjugated fractions are isolated by means of solid-phase extraction, using BE C18 and BE SAX cartridges in succession [23]. The taurine fraction is enzymatically hydrolysed according to a previously described method [24]. The final residue is treated with 1 ml volume of 0.01% (w/v) KOH methanolic solution (methanokwater 1 9, v/v) at 40°C by ultrasonication for 3 min. Then, 0.2 ml of the obtained suspension is derivatised as described below. BA content is determined in each sample by comparison with an appropriate standard solution. 

Used BA standards, CA, CDCA, DCA, UDCA, LCA, and their glycine- and taurine-conjugated forms are dissolved in methanol at a concentration of 1 mg/ml. 

This method allows a quantitative analysis of BAs present in biological fluids (free, and glycine- and taurine-conjugated forms) in a single chromatographic run, performed with an HPLC mass spectrometric system equipped with an electrospray interface [33]. 

Fig. 5.4.16 Typical room temperature nuclear magnetic resonance spectra of human BA and standards of glycine- and taurine-conjugated bile acid, (reprinted from [40])...

BAs are steroid compounds, hydroxyl-derivatives of 5-P-cholan-24-oic acid. BAs are the hnal products of the hepatic biotransformation of cholesterol and they are normally present in the bile as mixed micelles. They have different physicochemical properties according to the number, position, and orientation of their hydroxyl groups, and the type of conjugation with glycine and taurine, which forms the glyco- and tauro-derivatives. These factors influence their solubility, hydrophobicity, and detergent properties [35-37]. 

Bile acids (BA) are a group of acidic steroids synthesized in the liver from cholesterol. After conjugation with glycine or taurine, BA are secreted into the bile and enter the enterohepa-tic circi lation since they are efficiently reabsorbed by the intestine. ... 

The most prominent BA present in human are cholic acid (C), chenodeoxy-cholic acid (CDC), deoxycholic acid (DC), lithocholic acid (LC), and ursodeoxycholic acid (UDC), as derivatives of 5p-cholan-24-oic acid. Primarily they are present as glycine and taurine conjugates, with the conjugation occurring at carbon 24 of the structure. In addition to the above major BA, a wide array of minor components has been identified. 

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