Trihydroxy Bile Acids

Ions arising from the side chain are clearly seen and the fragmentation corresponds to that of fatty acid esters (46,47). The base peak in the spectrum of the bisnorcholic acid derivative is the side chain ion at mje 88, and the three-carbon end of the C27 acid derivative yields a prominent peak at mje 88. The peak corresponding to mje 154 in the C24 bile acids (/ g) is seen in the spectra of the derivatives of the C23-C27 acids. The ABCD and ABC ring ions are seen at mje 253 and 211, respectively. 

Fragmentation /j gives rise to a diagnostically very important peak. The fragmentation mechanism is not known. The peak is found at mIe 261 

The spectra of the silyl ethers also show a fragmentation indicating the presence of vicinal trimethylsiloxy groups A/-(2x90-1-89) The last 

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Currently available BAS include cholestyramine, colestipol and colesevelam hydrochloride (colestimide). Cholestyramine comprises a long-chain polymer of styrene with divinylbenzene trimethylbenzylammonium groups, whereas colestipol is a long-chain polymer of l-chloro-2,3-epoxypropane with diethylenetriamine. Colesevelam HCl is poly(allylamine hydrochloride) cross-linked with epichlorohydrin and alkylated with 1-bromodecane and 6-bromo-hexyl-trimethylammonium bromide. Bile-acid binding is enhanced and stabilised in the latter compound by long hydrophobic sidechains, increased density of primary amines, and quaternary amine sidechains. For this reason, colesevelam HCl exhibits increased affinity, specificity and capacity to bind bile acids compared with the other BAS. Colesevelam HCl also binds dihydroxy and trihydroxy bile acids with equal affinity, contrasting with cholestyramine and colestipol that preferentially bind dihydroxy bile acids (CDCA and deoxycholic acid). The latter BAS can lead to an imbalance towards trihydroxy bile acids and a more hydrophilic bile-acid pool. 

The epimerizalion of the 7a-hydroxyl group can occur either by intra- or interspecies mechanisms [16]. However, it is difficult to quantitatively assess the degree of 7-hydroxy epimerization in vivo because this transformation competes with the irreversible 7-dehydroxylation of bile acids (Section VI). 7a-HSDH activity has been reported in several genera of intestinal bacteria however, the most complete characterization of this enzyme has been carried out with the enzyme isolated from Escherichia coli [37] and Bacteroides sp. [29,38,39] (Table 2). Both enzymes used both free and conjugated bile acids as substrates, showed alkaline pH optima and lower values for dihydroxy than for trihydroxy bile acids. However, cell extracts prepared from Bacteriodes sp. contained both NAD- and NADP-depen-dent 7 -HSDH activities whereas, extracts from E. coli contained only an NAD-de-pendent enzyme activity. Additional studies showed that the two 7a-HSDH activities detected in Bacteriodes sp. differed in molecular weight, differential heat inactivation and Mn " requirement, suggesting the presence of two distinct enzymes [29]. 

No studies have compared the solubilization of a given fatty acid by different bile acids. Although trihydroxy bile acids have a lower saturation ratio than dihydroxy acids, it seems unlikely that this difference would be of physiological significance. 

Children with growth hormone deficiency produce only trihydroxy bile acids and have a low intestinal bile salt concentration (165). Growth hormone treatment restores these abnormalities and leads to improved intestinal absorption. A patient with acromegaly exhibited, after hypophysectomy with an adequate thyroid and adrenal hormone substitution, a markedly augmented bile acid synthesis (593 mg/day) which, however, was normal when related to the body weight (88). 

The quantitative analysis of the fecal bile acids of rats presents a special problem which does not need to be considered in comparable studies with most other species. The presence of appreciable quantities of muricholic acids, over 50% of the total with germfree feces, requires methods which will not cause destruction of these 3,6,7-trihydroxy bile acids if chromatographic techniques are used. 

It has been shown that the trifluoroacetates of 3,6,7-trihydroxy bile acids are subject to thermal decomposition in gas chromatographs (30). Oxidation of the bile acids to their keto derivatives and subsequent gas chromatography should also be avoided (31). In our laboratory, we have been unable to gas chromatograph any oxidized 3,6,7 bile acid methyl esters they are either destroyed or will not elute in a reasonable amount of time. 

A simpler practice to analyze bile acids is to use negative-ion FAB-MS [53,54]. A Sep-Pak Cig cartridge can be used to isolate bile acids (e.g., from urine sample) prior to FAB-MS analysis. Currently, ESI-MS has largely supplemented FAB-MS to analyze polar bile acids. The coupling of ESI with MS/MS provides an additional level of specificity. For example, neutral-loss scans for 36 and 54 Da can detect di- and trihydroxy bile acids. LC/ESI-MS can be used for the sensitive detection of bile acids in biological extracts. Recently, cholic acid, chenodeoxycholic acid, and deoxycholic acid have been detected in the cytoplasmic fraction from a rat brain [55]. 

Feature of 24-hydroxy-C27 tetrahydroxy-bile acid Base peaks in A in C27 trihydroxy-bile acid Feature of 24-hydroxy-C27 trihydroxy-bile acid Base peaks in A in C27 trihydroxy-bile acid Prominent in 22-hydroxy-CDCA Characteristic of 3,6,7-trihydroxy structures... 

The quantification of methyl cholanoates requires the resolving power of a QF-1 column for complex mixtures. Only little tailing with di- and trihydroxy bile acid methyl esters should be permitted. Under acceptable conditions the response relative to methyl deoxycholate is of the order 0.90-1.25 for mono- and disubstituted methyl cholanoates but may be as low as 0.50 for the trisubstituted compounds (18). Poor responses are probably due to column imperfections and not so much caused by the detection unit in the linear range. 

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... 

In substituted methyl cholanoates (Table I) loss of water or its equivalents (acetic acid, trifluoroacetic acid, trimethylsilanol, etc.) is pronounced. In gas chromatography-mass spectrometry this process is partly thermal, partly due to electron impact when a direct probe is used, thermal elimination can be avoided. Since the mechanisms of the two types of elimination are different (26) the spectra will differ to some extent. However, in work with biological materials, the complexity of the mixtures and the small amounts of bile acids available usually make it necessary to use the former method and to accept the thermal component in the fragmentation process. When methyl esters of di- and trihydroxy bile acids and their acetates or trifluoroacetates are analyzed by gas chromatography-mass spectrometry, a molecular ion is usually not seen. This is partly due to the high temperatures used. Trimethylsilyl ethers usually give a molecular ion peak but it may be quite small. 

Therefore, I shall concentrate on only those bile acids that have been reasonably well studied from a physicochemical point of view and which have some relation to physiology and biochemistry of living things. Because the specific physical characteristics of the bile acids and their alkaline metal salts vary considerably with the number of hydroxyl groups present on the steroid nucleus, I will present a fairly detailed description of the physicochemical properties of cholanic acid (no hydroxyl groups), monohydroxy, dihydroxy, and trihydroxy bile acids. Since the triketo bile acid (dehydrocholic acid) has been used widely as a choleretic, its properties will also be discussed. Unfortunately, many interesting bile acids and bile alcohols isolated from a variety of vertebrates (29-32) have not been studied physicochemical ly. However, knowing their molecular structure, many of the properties of these compounds can be deduced by comparison with the known properties of bile acids discussed in this chapter. 

The taurine conjugates of di- and trihydroxy bile acids are very strong acids and are very soluble down to pH = 1 (125-127). 

A major and striking physicochemical difference between lithocholic acid and the dihydroxy and trihydroxy bile acids is the insolubility of the sodium salts of the former (10, 45). Sodium salts of the common bile acids (taurine and glycine conjugates of cholic acid, deoxycholic acid, and che-nodeoxycholic acid) are very soluble in water and physiological saline, even at 0°C. The solubility of the ammonium, lithium, sodium, potassium, rubidium, and cesium salts of lithocholic acid (NH4L, LiL, NaL, RbL, CsL) have been studied in water as a function of temperature (45). 

Some thirty years ago Swell et al. showed that the rat intestinal mucosa possesses an enzymatic activity which catalyzes the formation of cholesterol esters[46,47]. The enzyme was called Cholesterol Esterase and its properties were similar to those of the pancreatic cholesterol esterase. Over the past three decades Vahouny and colleagues extensively studied the properties of this enzyme, its subcellular localization, the possible sources and finally they proceeded to the purification of the enzyme[5,26,28,48]. The authors hypothesize that the pancreas secretes subunits of cholesterol esterase. In the intestinal lumen the subunits, in presence of trihydroxy bile acids, aggregate to the active enz3me which catalyzes the hydrolysis of cholesterol esters from a micellar substrate and at the optimal pH of 6.6. The enz3one is then absorbed and localizes mostly in the supernatant of the mucosal cells here, at the optimal pH of 6.2, cholesterol esterase operates in reverse inducing the esterification of cholesterol and speeding up its... 

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