Bile, acid

The initial complications of the analysis of the bile acids are due to their natural occurrence as various biological conjugates (e.g., as glycine, taurine and various 

Formation of suitable derivatives from bile acids has also been a subject of numerous studies. Since the earlier procedures have been reviewed [314-316], a continuous interest in the subject of derivatization appears to indicate that there is still a need for improvement. The initial derivatization of the acidic function to form methyl esters seems to be almost universally employed, while the remaining functional groups (hydroxy and keto) can be converted to a number of derivatives. A number of earlier and more recent studies have endorsed silylation, although various types of acylation are also common [219]. Permethylation has also been advocated recently [22S], but no biological separations were demonstrated using this approach. 

The mixtures of bile acids isolated from biological materials can be exceedingly complex. A recent interest in capillary GC of these compounds [321,322,225,267] is thus justified. Interestingly, even a partial derivatization has been advocated [323] to increase resolution of various bile acids which are not adequately resolved when all polar groups are fully covered. A need for reliable identification and characterization techniques is reflected in the systematic investigations of chromatographic retention and mass-spectral studies of various bile acid derivatives [219,322,324,325]. 

Mass spectrometry has become an indispensable method for the analysis of bile acids by virtue of its power to identify, assign structure and quantify free or conjugated bile acids, either pure or in mixtures. It is useful not only to study the metabolism of bile acids but also for the detection and diagnosis of metabolic diseases. Indeed, numerous metabolic diseases resulting from an alteration of the conversion of cholesterol to bile acids have been described, including peroxisomal disorders resulting in a block of (3-oxidation of the lateral chain and other enzyme deficiencies interfering with the biochemistry of the side chain or the steroid nucleus. 

Before the advent of soft ionization techniques, the analysis of bile acids was long and tedious and needed large sample quantities. First, the bile acids had to be extracted from the biological fluid and separated by lipophilic ion exchange chromatography into four classes  

Collision-induced dissociation FAB/MS/MS traces of the taurine conjugates of 7a-hydroxy-3-oxochol-4-en-24-oic acid (top) and 7a, 12a-dihydro-3-oxochol-4-en-24-oic acid (bottom). Reproduced (modified) from Libert R., Hermans D., Draye J.P., Van Hoof F., Sokal E. and de Hoffmann E., Clin. Chem., 37, 2102-2110, 1991, with permission. 

The emergence of soft ionization techniques such as FAB, thermospray (TSP), APCI and ESI, combined with MS/MS, will considerably simplify these analysis. On a much smaller sample quantity, bile acids can be analysed without prior separation or derivati-zation. Soft ionization techniques are well suited for such polar, non-volatile thermolabile compounds. 

The FAB, TSP, APCI and ESI mass spectra in the positive ionization mode display the protonated molecular ion, generally accompanied by other adducts of the molecular species and fragments resulting from the loss of one or several water molecules originating from the ring hydroxyl groups. [300-302] As an acidic function is always present in these compounds, they also yield intense ions in the negative ion mode. [303-305] The spectra 

The elaboration of the sequence of steps required to convert cholesterol to bile acids in vivo has been largely due to the outstanding work of Bergstrom and his co-workers and has been reviewed by him (1955 1959 1960). The conversion 

Based on his examination of the bile salts of various species, Haslewood (1959) had concluded that an evolutionary pattern exists which is reflected in the pathway by which the cholesterol side chain is cleaved to yield the flve carbon acidic side chain of the bile acids. Thus, the older animals (shark) possess a side chain that has a 27 hydroxyl group, the reptiles mainly synthesize a 27 carboxylic acid and mammals a 25 carboxylic acid. Haslewood suggested that 3a, 7a, 12a-tri-hydroxycoprostanic acid might be an intermediate in cholic acid formation in 1952. 

From his studies in a number of mammahan species, Bergstrom (1960) concluded that the 7 a hydroxyl group of the bile acids is eliminated by the action of intestinal microorganisms during the course of enterohepatic circulation. Work with the rabbit, for instance, whose major bile acid is deoxychoflc acid has shown that this acid predominates because of the efficiency of the intestinal flora plus the inabihty of this animal to hydroxylate deoxychoflc acid. The rat, on the other hand, has a potent 7a hydroxylating enzyme. 

The in vivo work on bile acid formation has been augmented by work with liver homogenates and particulate fractions which has expanded the knowledge concerning the mechanisms involved. Chaikoff et al. (1952) had shown that large amounts of were formed after the administration of cholesterol-26-to 

When trihydroxycoprostanic acid labeled in the 4 position (prepared in the same manner as the 26-labeled acid) is incubated with rat liver mitochondria, labeled cholic acid is obtained. Danielsson had been carrying out parallel experiments in mouse liver mitochondria, and his work yielded valuable information concerning the pattern of hydroxylation of cholesterol. Cholesterol can be converted to 26 hydroxycholesterol and also to the 3/5, 7a, 26-triol. Danielsson also showed that liver mitochondrial preparations could convert 3 a, 7 a, 12 a trihydroxy coprostane to 3a, 7 a, 12 a, 26-tetrahydroxycoprostane, 3 a, 7 a, 12a-tri- 

BA are a group of compounds characterized by the steroid scaffolding with a carboxyl group located in the side chain. These compounds are the major catabolic products of cholesterol and facilitate either the excretion of bile lipids including cholesterol or the absorption of dietary lipids. 

XIC t +MRM (9 pali ) 331.997.3 amu (nom Sample a (3) ol OalaOS 04 3a05.wiH (Turpo Spiiy, Smoolhetf. Smoomea 

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. 

Hepato-biliary and intestinal diseases are marked by their increased concentration in plasma, urinary, and feces. Early diagnosis of many pathological conditions is often possible through individual separation and quantitation of BA. 

The analysis of BA has been always challenging due to their wide variety, lack of any volatility, very low concentration in biological samples, and the small structural differences between them, with several cases of isomeric forms. 

Bile is an important product released by the hepatocytes. It promotes the digestion of fats from food by emulsifying them in the small intestine (see p. 2770). The emulsifying components of bile, apart from phospholipids, mainly consist of bile acids and bile salts (see below). The bile also contains free cholesterol, which is excreted in this way (see p. 312). 

Cholic acid and chenodeoxycholic acid, known as the primary bile acids, are quantitatively the most important metabolites of cholesterol. After being biosynthesized, they are mostly activated with coenzyme A and then conjugated with glycine or the non-pro-teinogenic amino acid taurine (see p. 62). The acid amides formed in this way are known as conjugated bile acids or bile salts. They are even more amphipathic than the primary products. 

Deoxycholic acid and lithocholic acid are only formed in the intestine by enzymatic 

Bile salts are exclusively synthesized in the liver (see A). The slowest step in their biosynthesis is hydroxylation at position 7 by a 7-a-hydroxylase. Cholic acid and other bile acids inhibit this reaction (end-product inhibition). In this way, the bile acids present in the liver regulate the rate of cholesterol utilization. 

Before leaving the liver, a large proportion of the bile acids are activated with CoA and then conjugated with the amino acids g/ycine or taurine (2 cf A). In this way, cholic acid gives rise to glycocholic acid and taurocholic acid. The liver bile secreted by the liver becomes denser in the gallbladder as a result of the removal of water (bladder bile 3). 

The liver secretes a elear, golden yellow viscous liquid known as bile . It is stored in gall bladder and is solely useful for the digestive system. It mainly consists of the inorganie ions like HCO3, Cl Na , K, etc., in addition to organic compoimds such as bile aeids, bile pigments, liquid fatty acids and cholesterol. Cholic Acid Deoxycholic Acid Chenodeoxycholic Acid. 

The bile acids are usually present as the salt of amide with either glycine or taurine, for instance sodium glycocholate (glycine + cholic acid), and sodium taurocholate (taurine + cholic acid). 

In all twelve natural bile aeids have been identified and eharacterised duly. Of these the most abundant bile aeids in human bile are cholic acid (26-60% of total bile acids) deoxycholic acid (5-25%), and chenodeoxycholic acids (30-35%), whose struetures and chemical names are stated below  

The bile acids may be isolated from the bile by eleaving the peptide linkage present in them by hydrolysis with alkali. From the resulting solution the bile acids are eonveniently isolated either by crystallization from organic solvent or by treating the ethereal solution of the aeids with various concentration of hydrochloric acid, for instance the trihydroxy, dihydroxy and the monohydroxy acids may be isolated by treating the ethereal solution with 15%, 25% and eoneentrated hydroehloric acid respectively. 

Until recently, desoxycholic acid was the only starting material for the synthesis of cortisone, and its utilization involved three distinct features  

Desoxycholic acid (III) is commercially available, but its 7-hydroxy analog, cholic acid (I), is much more abundant in animal bile. One of the best conversions is due to Fieser and Rajagopalan (1949), who oxidized cholic acid (I) with N-bromosuccinimide to the 7-keto derivative (II), which without purification is reduced in 68% over-all yield by the Huang-Minlon modification of the Wolff-Kishner method to desoxycholic acid (III). An alternative reduction, devised by Hirschmann et al. (1951), involves the catalytic hydrogenolysis of the enol acetate of II, but the yield is inferior. 

A -3a-Hydroxy-12-ketocholenic acid (VI) represents another important starting material for cortisone syntheses (Fig. 5), and two useful methods for its preparation are given in Fig. 1. The first method (Schwenk and Stahl, 1947 McKenzie et al., 1948) involves a preferential acylation at C-3 of desoxycholic acid (III), followed by chromium trioxide oxidation 

One of the simplest and most efiicient processes for accomplishing the shift of the C-12 oxygen function to C-11 is due to Gallagher. Methyl desoxycholate (Fig. 1) upon partial acetylation at C-3 and oxidation at C-12 leads readily to methyl 3a-acetoxy-12-ketocholanate (I). Bromination 

Synthesis of Cortisone from 11-Ketolithocholic Acid Figs, 8 and 4) 

Knuppen, P. Ball, O. Haupt, and H. Breuer, Z. physiol. Chem., I97Z 353, 565 T. Nambara, 

Kanayama. Chem. and Pharm. Bull. (Japan), 1972,20,2235. 

Manson, L. Nocke-Fink, J.-A. Gustafsson, and C. H. L. Shackleton, Clinica Chim. Acta, 1972,38,45. 

Yamasaki. Yonaga Acta Medico. 1971.15,171 B. Croizat, M. Lambiotte. N. Simonet-Thierry, and J. Kande, Am. Nutr. Aliment., 1971 25,203. 

Several experiments have been reportedon the biosynthesis of bile acids in rats administered [l- Hj]ethanol for labelling of the NADH and NADPH pools. The fact that deuterium was present in the 3P-position but not in the Sa-position of 3a-hydroxy-5a-cholanoic acid formed from 3-oxochol-4-en-24-oic acid indicated that different coenzyme pools are utilized in the reductions of the oxo-group and the double bond. This is in agreement with other observations.It is interesting that unidentified unsaturated monohydroxy bile acids have been isolated from patients with cholestatic liver disease.  

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Mammalian bile contains sodium salts of conjugated bile acids, e.g. glycocholic acid and taurocholic acid, in which cholic acid is combined (amide linkage) with glycine and taurine respectively. 

CJH4O5, H02CCH(0H)C02H. Colourless crystals with IH O lost at 60 C. M.p. IhO C (decomp.). Prepared by heating dinitrotartaric acid in aqueous alcohol, taurine, aminoethylsulpbonic acid, C2H7NO3S, NHj CHj CH SOjH. Crystallizes in columns, decomposing at 317 C. In combination with cholic acid it forms one of the bile acids. It is formed in the liver from cysteine. 

M. C. Carey, in Enterohepatic Circulation of Bile Acids and Sterol Metabolism, G. Paumgartner, ed., MTP Press, Lancaster, Boston, 1984. 

A significant fraction of the body s cholesterol is used to form bile acids Oxidation m the liver removes a portion of the CsHi7 side chain and additional hydroxyl groups are intro duced at various positions on the steroid nucleus Cholic acid is the most abundant of the bile acids In the form of certain amide derivatives called bile salts, of which sodium tau rocholate is one example bile acids act as emulsifying agents to aid the digestion of fats... 

The outer layer or cortex of the adrenal gland is the source of a large group of sub stances known as corticosteroids Like the bile acids they are derived from cholesterol by oxidation with cleavage of a portion of the alkyl substituent on the D ring Cortisol IS the most abundant of the corticosteroids but cortisone is probably the best known Cortisone is commonly prescribed as an antiinflammatory drug especially m the treat ment of rheumatoid arthritis... 

Bile acids (Section 26 13) Steroid derivatives biosynthesized in the liver that aid digestion by emulsifying fats Bimolecular (Section 4 8) A process in which two particles re act in the same elementary step Biological isoprene unit (Section 26 8) Isopentenyl pyrophos phate the biological precursor to terpenes and steroids... 

Cholesterol (Section 26 11) The most abundant steroid in am mals and the biological precursor to other naturally occur ring steroids including the bile acids sex hormones and corticosteroids... 

Biguanide salts Biimidazole dimer Biisobutyryl [4388-87-8] Bilarcil Bile acids... 

Diarrhea is a common problem that is usually self-limiting and of short duration. Increased accumulations of small intestinal and colonic contents are known to be responsible for producing diarrhea. The former may be caused by increased intestinal secretion which may be enterotoxin-induced, eg, cholera and E. col] or hormone and dmg-induced, eg, caffeine, prostaglandins, and laxatives decreased intestinal absorption because of decreased mucosal surface area, mucosal disease, eg, tropical spme, or osmotic deficiency, eg, disaccharidase or lactase deficiency and rapid transit of contents. An increased accumulation of colonic content may be linked to increased colonic secretion owing to hydroxy fatty acid or bile acids, and exudation, eg, inflammatory bowel disease or amebiasis decreased colonic absorption caused by decreased surface area, mucosal disease, and osmotic factors and rapid transit, eg, irritable bowel syndrome. 

Clinical Analysis. A wide range of clinically important substances can be detected and quantitated using chemiluminescence or bioluminescence methods. Coupled enzyme assay protocols permit the measurement of kinase, dehydrogenase, and oxidases or the substrates of these enzymes as exemplified by reactions of glucose, creatine phosphate, and bile acid in the following ... 

Experimental procedures have been described in which the desired reactions have been carried out either by whole microbial cells or by enzymes (1—3). These involve carbohydrates (qv) (4,5) steroids (qv), sterols, and bile acids (6—11) nonsteroid cycHc compounds (12) ahcycHc and alkane hydroxylations (13—16) alkaloids (7,17,18) various pharmaceuticals (qv) (19—21), including antibiotics (19—24) and miscellaneous natural products (25—27). Reviews of the microbial oxidation of aUphatic and aromatic hydrocarbons (qv) (28), monoterpenes (29,30), pesticides (qv) (31,32), lignin (qv) (33,34), flavors and fragrances (35), and other organic molecules (8,12,36,37) have been pubflshed (see Enzyp applications, industrial Enzyt s in organic synthesis Elavors AND spices). 

Initial steroid research involved isolation of sterols and bile acids from natural sources. DeFourcroy is generally credited with the discovery of cholesterol [57-88-5] (2) in 1789 (3). In 1848, choHc acid [81-25-4] (3) was isolated from the saponification of ox bile and its elementary composition deterrnined as... 

Although many sterols and bile acids were isolated in the nineteenth century, it was not until the twentieth century that the stmcture of the steroid nucleus was first elucidated (5). X-ray crystallographic data first suggested that the steroid nucleus was a thin, lath-shaped stmcture (6). This perhydro-l,2-cyclopentenophenanthrene ring system was eventually confirmed by the identification of the Diels hydrocarbon [549-88-2] (4) and by the total synthesis of equilenin [517-09-9] (5) (7). 

Pregniines. In 1944, Sarrett completed the first partial synthesis of cortisone (172). Like many of the early syntheses of corticosteroids, Sarrett began with the a bile acid, deoxychoHc acid (14). Because bile acids are isolated from animal sources, their supply is by necessity limited (173). Following these early syntheses, several improvements and innovations have resulted in a number of industrial syntheses of cortisol and other corticosteroids. 

H. Van BeUe, Cholesterol, Bile Acids, and Atherosclerosis, North-HoUand Publishing Co., Amsterdam, the Netherlands, 1965. 

G. Paumgartner, A. Stiehl, and W. Gerok, eds.. Bile Acids and Cholesterol in Health and Disease, MTP Press Ltd, Boston, Mass., 1983. 

Bile Acid Sequestrants. The bile acid binding resins, colestipol [26658424] and cholestyramine, ate also effective in controlling semm cholesterol levels (150). Cholestyramine, a polymer having mol wt > ICf, is an anion-exchange resin. It is not absorbed in the gastrointestinal tract, is not affected by digestive enzymes, and is taken orally after being suspended in water (151). 

Molecular Interactions. Various polysaccharides readily associate with other substances, including bile acids and cholesterol, proteins, small organic molecules, inorganic salts, and ions. Anionic polysaccharides form salts and chelate complexes with cations some neutral polysaccharides form complexes with inorganic salts and some interactions are stmcture specific. Starch amylose and the linear branches of amylopectin form inclusion complexes with several classes of polar molecules, including fatty acids, glycerides, alcohols, esters, ketones, and iodine/iodide. The absorbed molecule occupies the cavity of the amylose helix, which has the capacity to expand somewhat to accommodate larger molecules. The starch—Hpid complex is important in food systems. Whether similar inclusion complexes can form with any of the dietary fiber components is not known. 

Dietary fiber and fiber-rich food fractions bind bile acids and bile salts in vitro. This interaction is more pronounced for the lignin component. 

A number of steroids have been regioselectively acylated ia a similar manner (99,104). Chromobactenum viscosum hpase esterifies 5a-androstane-3P,17P-diol [571-20-0] (75) with 2,2,2-triduoroethyl butyrate ia acetone with high selectivity. The hpase acylates exclusively the hydroxy group ia the 3-position giving the 3P-(monobutyryl ester) of (75) ia 83% yield. In contrast, bacillus subtilis protease (subtihsia) displays a marked preference for the C-17 hydroxyl. Candida iylindracea]i 2Lse (CCL) suspended ia anhydrous benzene regioselectively acylates the 3a-hydroxyl group of several bile acid derivatives (104). 

It is important to note that diet is a complex mixture that contain compounds with varying activity. Chemical stimulators of colon cancer growth include bile acids, 1,2-diglycerides and prostaglandins which stem from consumption of fat. In contrast, fruits and vegetables contain substances such as carotenoids, flavonoids and fibre, which may inhibit cancer cell growth, and the risk of colon cancer appears to be mirrored by the ratio of plant sterols to cholesterol in the... 

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