Bile acids analyses

N—55, 45 GORD patients, 10 controls. HPLC for bile acid analysis. 

Cool to room temperature, add 10 ml hexane or petroleum ether, tighten caps, and shake vigorously for 15 sec. Centrifuge at 1000 7 for 10 min to separate phases and remove the organic layer (top) to a fresh tube or scintillation vial. Repeat the extraction twice and combine organic layers. Save the aqueous layer (bottom) in the original tube for bile acid analysis. 

As well as the enzyme 3a-hydroxysteroid dehydrogenase, a 7a-enzyme has been isolated from E. coli and a 12a enzyme from a strain of Clostridium (Ml). These group-specific enzymes can be used to measure the amounts and ratios of the three main bile acids (cholic, chenodeoxycholic, and deoxy-cholic acids) in bile specimens. Bioluminescent assays suitable for serum bile acid analysis have been described using 7a-hydroxysteroid dehydrogenase (R6) and 12a-hydroxysteroid dehydn enase (S12), as well as for the 3a enzyme (S13, S44). 

It is still too early to tell whether serum bile acid determinations will be added to the currently available combination of liver function tests or replace individual tests, such as the measurement of bilirubin (H19). In correctly diagnosing patients with histologically defined liver disease, serum bile acids appear to slightly improve the results from conventional liver tests, if used in combination with these tests (F3). Perhaps when sensitive analytical methods such as bioluminescence, which can be applied to serum bile acid analysis, become available and established in diagnostic laboratories, serious consideration will be given to routine measurement of serum bile acid levels to... 

Two other areas which are being explored and involve serum bile acid analysis deserve mention. While many studies have concentrated on their diagnostic value, serum bile acids may have potential for predicting the prognosis of chronic liver disease. Monroe et al. have found that the degree of serum bile acid elevation correlated with histological severity of liver dami e in chronic hepatitis patients (M31) and serial bile acid determina-... 

Setchell, K. D. R., and Matsui, A., Serum bile acid analysis. Clin. Chim. Acta 127,1-17 (1983). 

W6. Whiting, M. J., and Watts, J. McK., Prediction ofthe bile acid composition of bile fit>m serum bile acid analysis during gallstone dissolution therapy. Gastroenterology 78, 280-225 (1980). 

Several reviews covering different aspects of bile acid analysis have appeared in recent years (1-15). These articles cover a wide range of techniques with particular emphasis on thin-layer and gas chromatography. In this chapter we will try to present established methods for bile acid analysis as they can be combined to form a complete analytical procedure, including extraction, purification, identification, and quantitation. It has not been possible to include all methods and the various modifications of standard procedures that have been published. References to most of these can be found in the reviews (1-15). Early development in the field is described in Sobotka s books on bile acids and steroids (16, 17). Whenever equally efficient or similar methods exist we have generally preferred to describe those of which we have personal experience. Many of the techniques used for bile acids are similar to those used in steroid analysis and advance in this area is highly relevant for workers in the bile acid field. 

Reversed phase partition conditions are established with alkylated Sephadex LH-20 containing 50% (w/w) of Cji-Cj4 alkyl chains, when solvents such as methanol-water-ethylene chloride, 70 30 10, are used. With this system 3a-hydroxy-, 3/3-hydroxy-, and 3-keto-5p-cholanoic acids are completely separated in this order (65). The importance of liquid-gel chromatography in bile acid analysis is likely to increase during the next few years. 

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. 

Bile acid analysis consists of sample workup and final determination. Developments are necessary in both parts of the analysis. Initial progress... 

Future development is likely to include improvements in column liquid-liquid chromatography—new column packing materials and new detection systems. Stationary phases consisting of specific groups covalently bound to inert matrices are being developed in many laboratories for use in liquid-liquid as well as gas-liquid chromatography. Such phases might be made for use in bile acid analysis. 

Bile Acid Ethyl Esters. Their Infrequent Formation During Routine Bile Acid Analysis and Identification by Gas Chromatography and Mass Spectrometry... 

BIOCHEMICAL METHODS FOR SERUM BILE ACID ANALYSIS... 

Shimoyama, Fecal bile acid analysis in healthy Japanese subjects using lipophilic anion exchanger, capillary column gas chromatography and mass spectrometry, Gastroenterologia Japonica, 16 363 (1983). 

Biochemical Methods for Serum Bile Acid Analysis.. A. Roda, S. Girotti, P. Filippetti,... 

In diagnoses of some rare conditions, such as bile acid synthetic defects, MS can also be utilized. Nowadays it is possible to screen and rapidly diagnose potential or real inborn errors in bile acid synthesis from urinary bile acid analysis by means of MS. Specific mutations in the genes that encode the enzymes responsible for bile acid synthesis can be identified by molecular techniques. Of the seven known genetic defects that cause progressive cholestatic Uver disease, syndromes of fat-soluble vitamin malabsorption, and neurological disease, six have been properly characterized [16]. 

Budai, K. Javitt, N. B. Bile acid analysis in biological fluids a novel approach. J. Lipid Res. 1997, 38, 1906-1912. 

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