Bile acids in biological materials

B35. Bruusgaard, A., Quantitative determination of the major 3-hydroxy bile acids in biological material after thin-layer chromatographic separation. Clin. Chkn. Acta 28, 495-504... 

E2. Eneroth, F., and SjOvall, J., Extraction, purification and diromatographic analysis of bile acids in biological materials. In The Bile Acids-Chemistry, Physiology and Metabolism (P. P. Nair and D. Kritchevsky, eds.), Vol. 1, pp. 121-168. Plenum, New York, 1971. 

AND CHROMATOGRAPHIC ANALYSIS OF BILE ACIDS IN BIOLOGICAL MATERIALS ... 

Sakaguchi, K., Katayama, K., Tsutsumi, J., and Kawabe, K. Determination Method of Bile Acids in Biological Materials by Mass Fragmentography Yakugaku Zasshi 99(4) 421-431 (1979) CA 91 71116g... 

Significance of Bacterial Steroid degradation for the Etiology of Large Bowel Cancer. III. Possib= ilites for the Determination of Bile Acids in Biological Material... 

Radioimmunoassay. Antibodies can be produced against bile acid-protein complexes - usually bile acid-bovine serum albumin complex - and used for radioimmunoassay of that bile acid in usual manner (cf. 1). Each bile acid should have its own antibody so that the method is suitable for the quantitation of individual bile acids provided the antibodies are specific enough. One of the major problems actually is the cross-reactivity of antibodies and difficulties in the determination of total bile acids in biological materials. Thus, for the fec il bile acids with mainly bacterial transformation products (see Fig. 1) and to some extent for the urinary bile acids, especially under diseased conditions, this is quite Impossible. 

Although this class of substances has observed a fiftieth anniversary in man s knowledge of bile acids, the subject has not sparked biochemical interest until recent times, when, aided by modern techniques, the biochemist has been able to study smaller quantities of materials at the subcellular level. It seems likely that this is the level where new knowledge of the biological importance of alio bile acids will be generated. Where there are current explanations for the roles of the 5/3 bile acids in nature, these answers have not yet appeared for the allo-acids, partially because of the newness of the subject and the paucity of materials. Until the role of the allo-acids in nature is fully detailed, investigations in this area will continue. 

The first step should preferably lead to a group separation of bile acids and to elimination of non-bile acid contaminants. In quantitative work it may be useful to add—at this stage or earlier—labeled compounds, which permit an estimation of recovery of bile acids of different polarities. Tauro-cholic and 3-ketocholanoic acids constitute suitable extremes in polarity. If specific bile acids are to be analyzed, one may add a suitable internal standard to the biological material. Thus, Rooversc/ r/. (36) used nordeoxy-cholic acid for gas chromatographic determination of bile acids in feces and plasma. 

Several examples of the applications of polarography in these fields have been already mentioned in Chapters VI and VII, viz. determinations of benzene, toluene, naphthalene and phenols in the atmosphere, breath, blood or urine, of amino acids (with particular interest to tyrosine, tryptophane, phenylalanine, histidine and histamine), of ketoacids, ketosteroids, carbon disulphide in air and blood, ethanol, acetoin, sugars and morphine in blood, of lactic acid, mandelic acid in bile and urine, adrenaline and thyroxine in iodinated proteins and last, but not least, of thiol compounds, both soluble and bound in biological materials. A few further examples will be given here. 

Many additional steroid compounds are encountered in relatively complex mixtures. An increasing use of GC for the separation of biological sterols has been noticed during the last decade. The materials of interest may include bacteria, algae, various plants, marine animals, mammalian tissues, etc. Various dietary aspects of sterols and their metabolites, including bile acids, have recently been studied to a large extent. 

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]. 

The primary source of cholesterol is the liver. After cholesterol is produced, the liver packages cholesterol molecules with proteins in units known as lipoproteins, which are then distributed throughout the body by way of the bloodstream. Some cholesterol is deposited in cells, where it is used as the raw material in the synthesis of a number of biologically essential compounds, such as vitamin D3, steroids (such as estrogen and testosterone), and the bile acids, used by the body to digest foods. Excess cholesterol not needed by cells remains in the bloodstream until it is returned to the liver. 

Shackleton, C. H., Merdinck, J., and Lawson, A. M. 1990. Steroid and Bile Acid Analyzes. In Mass Spectrometry of Biological Materials, ed. C. N. McEwen and B. S. Larsen, 297-377. New York Marcel Dekker. 

Cholesterol and its fatty acid esters are important components of nerve and brain cells and are precnrsors of the biological materials snch as bile acids and steroid hormones. Accumulation of cholesterol in blood leads to fatal diseases such as arteriosclerosis, cerebral thrombosis and coronary diseases. Kajiya and co-workers immobilised cholesterol oxidase (ChOx) and ferrocene carboxylate in PPy electrochemically to describe the sensitivity of the resulting films [188]. The response was proportional to the cholesterol concentration up to 0.05 mM. It has been demonstrated that ferrocene attached to polymer chains can mediate electron transfer from horseradish peroxidase (HRP) to a conventional electrode surface [189]. In the case of immobilised HRP and ChOx the sensor yields 0.35 i,A to 10 mM of cholesterol whereas 3 pA was obtained in the case of free ChOx. It was therefore suggested that the sensor response is limited by the interfacial transport or reaction rate of H2O2. The sensor response was also found to be independent of the applied potential between -100 and 100 mV. 

In work with crude biological bile acid mixtures it has proved advantageous to make an initial separation with a phase system of type F followed by rechromatography of material eluted with the solvent front in phase systems of the C or D type. The only drawback with these latter systems is that isooctanol and Az-butanol are difficult to evaporate. 

TLC has been used for quantitative bile acid analysis by several authors. This was discussed partly in section III.A.4. The main alternative methods are based on spectrophotometric determination before or after elution (11, 51-53, 88-91), densitometric or fluorimetric recordings (54, 92), and enzymatic determination after elution (50, 93-94). The major problem with use of colorimetric methods in work with biological materials is that of specificity (as evidenced by the number of method modifications). Although it may be difficult to elute bile acids from the adsorbent, satisfactory methods are now available (see I11.A.4) and it is likely that the methods based on elution and determination with 3a-hydroxysteroid dehydrogenase give the most reliable results. 

In addition to the anion exchangers, cation-exchange resins can also be usefully employed in bile acid work. Thus columns of a strong cation-exchange resin (Dowex-50, 2% cross-linked, 50-100 mesh, J.T. Baker Chemical Co.) have been used in the hydrogen form to purify extracts of bile acids prepared from biological materials (2, 30). Prior to use the resin is extracted... 

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. 

HPLC. Separation of complex bile acid mixtures with HPLC appears to be quite successful from many biological materials (cf. 1). However, the simultaneous presence of free bile acids, perhaps with a wide variety of transformation products, and taurine- and glycineconjugated and sulphated bile acids forms such a mixture that is not separated totally but require preliminary group isolation, e.g. with TLC or ionexchange chromatography. Another problem of HPLC is the lack of specified detection of separated components. Ultraviolet spectrophotometry, directly or after derivatization, differential refractometry and 3a-HSD are most commonly used detection methods. Any masking of 3a-OH results in underestimation of effluent bile acids. 

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