Free and glycine conjugated

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. 

Effect of pH. The effect of pH on the deoxycholate series of bile salts in 0.15N NaCl at 20° and 36°C. is summarized in Figure 8. Solutions of free and glycine conjugated bile salts were studied between pH 9.2 and their precipitation pH (NaDC == 6.95, NaGDC = 4.9). Taurine conjugates were studied between pH 10 and 1.6. 

Fig. 7. Potentiometric titration curves of free and glycine (G)-conjugated ursodeoxycholic (UDC) and chenodeoxycholic (CDC) acids. Inset in D represents typical titration curve for free and glycine-conjugated bile acids with notations as explained in the text. The arrows on the GCDC curves indicate the points of bile acid precipitation. (From ref. 35 with permission.)...

If there is reason to suspect the presence of several salt forms, one should convert the bile acids into only one form before attempting a chromatography in neutral organic solvents. This can be done with the cation exchanger Amberlyst A-15 [free and glycine-conjugated bile acids can be extracted with ethyl acetate from an acidified water solution but this cannot be done with... 

When the separations of the acids within the free and glycine conjugated groups are considered, it is seen that on both columns and thin layers, the cholic acid derivatives are eluted ahead of the derivatives of the deoxycholic and chenodeoxycholic acids, and presumably other dihydroxy bile acids. The derivatives of the lithocholic acid are eluted last. Complete separations of the mono-, di-, and trihydroxy bile acids are not realized even on the thin-layer plates of ion exchangers, and there is no discernible resolution of the various taurine conjugates. This order of elution of the bile acids is opposite to that expected on the basis of their pK values (Table II). Free cholic acid (pK 5.29) would have been expected to be retained longer than the dihydroxy acids (pK 6.18-6.29) which should have been retained... 

Examples of the titration curves for a pure bile salt (NaC) and pure conjugated bile salt solutions (NaGC and NaTC) are shown in Fig. 24. The curves for sodium cholate and sodium glycocholate are similar to the ideal curve (Fig. 21), but since sodium taurocholate is the salt of a strong acid and remains completely ionized, the solution remains clear throughout the titration and therefore neither the supersaturation nor the precipitation characteristic of the free and glycine-conjugated bile salts takes place. 

Okuyama, S. Uemura, D. Hirata, Y. The improved method of high performance liquid chromatographic separation of individual bile acids free and glycine-conjugated bile acids. Chem. Lett. 1979,461 62. 

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

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. 

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... 

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]. 

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])...

The strong-base anion-exchange paper has been employed (38) in the hydroxide form for the separation of free and taurine-conjugated bile acids with diisobutyl ketone-acetic acid-water (4 5 1, v/v/v) or chloroform-methanol-ammonia (4 1 0.1, v/v/v) as developing solvents. It would be anticipated that these systems would also be capable of resolving glycine-and taurine-conjugated bile acids. 

The aggregation number of the micelle (number of molecules of bile salt per micelle) as a function of the concentration of NaCl is given for free bile salts (Fig. 42), taurine conjugates (Fig. 43), and glycine conjugates (Fig. 44), respectively, at 20 °C and pH values from 8 to 9. 

Table 1.1 lists some of the characteristics of the more common bile acids, which are divided into 3 main classes free bile acids, glycine and taurine conjugates. 

Other drugs are metabolised by Phase II synthetic reactions, catalysed typically by non-microsomal enzymes. Processes include acetylation, sulphation, glycine conjugation and methylation. Phase II reactions may be affected less frequently by ageing. Thus according to some studies, the elimination of isoniazid, rifampicin (rifampin), paracetamol (acetaminophen), valproic acid, salicylate, indomethacin, lorazepam, oxazepam, and temazepam is not altered with age. However, other studies have demonstrated a reduction in metabolism of lorazepam, paracetamol (acetaminophen), ketoprofen, naproxen, morphine, free valproic acid, and salicylate, indicating that the effect of age on conjugation reactions is variable. 

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. 

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