High-performance liquid chromatographic-electrospray mass spectrometric analysis of bile acids in biological fluids

The prevention of the hepatotoxic effects produced by intravenous infusion of taurochenodeoxycholic acid (TCDCA) by coinfusion with taurohyodeoxycholic acid (THDCA) was evaluated in bile fistula rats; the hepatoprotective effects of the latter were also compared with those of tauroursodeoxycholic acid (TUDCA). Rats infused with TCDCA at a dose of 8 mol/min/kg showed reduced bile flow and calcium secretion, as well as increased biliary release of alkaline phosphatase (AP) and lactate dehydrogenase (LDH). This was associated with a very low biliary secretion rate of TCDCA (Ϸ1 mol/min/kg). Simultaneous infusion of THDCA or TUDCA at the same dose preserved bile flow and almost totally abolished the pathological leakage of the two enzymes into bile. The effect was slightly more potent for THDCA. The maximum secretion rate of TCDCA increased to the highest value (8 mol/min/kg) when coinfused with either of the two hepatoprotective bile acids (BA), which were efficiently and completely secreted in the bile, without metabolism. Calcium output was also restored and phospholipid (PL) secretion increased with respect to the control saline infusion. This increase was higher in the THDCA study. These data show that THDCA is highly effective in the prevention of hepatotoxicity induced by intravenous infusion of TCDCA by facilitating its biliary secretion and reducing its hepatic residence time; this was associated with selective stimulation of PL biliary secretion. (HEPATOLOGY 1998;27:520-525.)Taurohyodeoxycholic acid (THDCA) is the taurineamidated form of hyodeoxycholic acid, a natural bile acid present in the pig, other animal species, and also in humans. [1][2][3][4] The steroid structure is characterized by the presence of two hydroxy groups, 3␣ and 6␣, the latter of which is equatorial and oriented toward the back of the steroid moiety. As a result, this molecule is less detergent than the other dihydroxy bile acid (BA), such as chenodeoxycholic acid (CDCA) (3␣, 7␣) or its 3␣, 6␤ dihydroxy epimer. The hydrophobic-hydrophilic balance of THDCA resembles that of tauroursodeoxycholic acid (TUDCA), with which it also shares similar orientation of the 7␤ hydroxy group with respect to the 6␣ hydroxyl. The physicochemical properties of this molecule are quite similar to those of TUDCA in terms of lipophilicity and critical micellar concentration (CMC). 5,6 Because of its relatively high hydrophilicity, THDCA has been proposed for use as an alternative to ursodeoxycholic acid (UDCA) or TUDCA for the treatment of cholesterol gallstone dissolution. [7][8][9][10] Recent studies have reported its activity as a cholesterol-dissolving agent, both in vitro and in vivo, and this has been attributed to its ability to selectively stimulate biliary phospholipid secretion. 11 Moreover, hyodeoxycholic acid is a secondary BA derived from hyocholic acid by intestinal bacterial 7␣ dehydroxylation, and thus, it is quite stable to intestinal bacteria; this is a potential advantage compared with UDCA that can undergo either 7␤ dehydroxylation or 7␣...

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“…A mass spectrum of each separated BA was thus obtained, with a direct reading of their molecular weights. 20 …”

Section: Methodsmentioning

confidence: 99%

Taurohyodeoxycholic acid protects against taurochenodeoxycholic acid-induced cholestasis in the rat

et al. 1998

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“…HPLC with UV detection is too insensitive for analysis of serum bile acids unless large volumes of serum are used ( 204 ). One solution to the nondetection of unconjugated bile acids and bile alcohol sulfates is to use a light scattering detector that responds approximately equally to all bile acids ( 205 ). A second approach, useful for unconjugated bile acids, is to couple the bile acid to a chromophore such as the phenylacyl group ( 206 ).…”

Section: Enzymatic and Competitive Binding Assaysmentioning

confidence: 99%

Key discoveries in bile acid chemistry and biology and their clinical applications: history of the last eight decades

Hofmann

Hagey

2014

Journal of Lipid Research

304

284

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conceptions are rare, and provocative judgments stand the test of time. Let us also hope that this effort will be both useful and entertaining. Each of the topics discussed merits at least a full length article, so there will be, of necessity in many instances, consideration of only what we perceive to be the highlights. THE EBB AND FLOW OF MEDICAL INTEREST IN BILE ACIDSAfter the elucidation of the true chemical structure of bile acids in 1932 (see below), there was little interest in bile acids in the Western world. One exception to this statement was the laboratory of Siegfried Thannhauser, who wrote the fi rst textbook of metabolic biochemistry in Germany. He studied cholesterol and bile acid balance in the biliary fi stula dog ( 1 ). During this time, bile acids were sold as liver tonics and laxatives, but there were no placebo-controlled studies showing effi cacy. Indeed, bile acids were considered by the medical profession to have no useful therapeutic properties. The tri-oxo derivative of cholic acid (called "dehydrocholic acid") was known to induce bile fl ow in animals ( 2 ), and was occasionally used to stimulate bile fl ow in patients; but again there were no controlled studies showing effi cacy in hepatobiliary disease.

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“…In this section, we have concentrated on negative-ion ESI-MS and ESI-MS/MS; however, with this form of ionization, unconjugated bile acids tend to give a lower response than either aminoacyl amidated or sulfuric acid conjugated bile acids ( 13,38 ). This is also true of unconjugated bile alcohols, and these molecules may be better analyzed by GLC-MS following appropriate derivatization (see below).…”

Section: Whether Ms/ms Is Performed With High-or Low-energy Cid Isomementioning

confidence: 99%

“…Problems with HPLC have been limitations in detector sensitivity and specifi city. These problems were greatly reduced by the introduction of soft ionization methods, particularly electrospray ionization (ESI), permitting the use of mass spectrometers as detectors in HPLC also ( 13 ). Recent advances in bile acid analysis have been made primarily in HPLC-MS and this is the fi eld in which much of the future improvements in speed, sensitivity, and simplicity in bile acid analysis can be expected.…”

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Bile acids: analysis in biological fluids and tissues

Griffiths

Sjövall

2010

Journal of Lipid Research

169

146

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in lower vertebrates. Metabolism of cholesterol to bile acids/bile alcohols is of major importance for the maintenance and regulation of cholesterol homeostasis. Depending on their structure, bile acids/bile alcohols can be ligands to nuclear receptors with different regulatory functions in animals, including even Caenorhabditis elegans ( 1 ). Bile acids can be formed in some marine microorganisms ( 2 ). These fi ndings have increased interest in methods for specifi c and sensitive analysis of bile acids.Conjugated forms of bile acids/bile alcohols in bile (bile salts) are essential participants in the absorption of lipids and lipid soluble compounds. Studies of this function, bile secretion, enterohepatic circulation, levels in blood, urine, and feces required methods for analysis of bile acids. In 1844, M. von Pettenkofer described his classical color reaction between cholic acid(s) and sugar in conc. sulfuric acid. During the subsequent 100 years, the reaction was modifi ed in numerous ways. This period is described by Sobotka ( 3, 4 ), who pointed out the discrepancies between the poor quality of the analytical methods of his days and the better understanding of bile acid physiology and pathology. He made the following remarks, well worth considering today:"The value of a method lies not only in its specifi city but also in the ease with which interfering factors may be specifi cally removed. To test the usefulness of a method, it is insuffi cient to demonstrate that solutions of the pure substance yield correct results and that known amounts of the substance, when added to biological material, may be completely recovered. It is absolutely necessary to prove that the analytical reaction given by the biological material or extract is due to the substance for which one tests. The unequivocal identifi cation of this substance, in amounts consistent with the analytical data, should alAbstract The formation of bile acids/bile alcohols is of major importance for the maintenance of cholesterol homeostasis. Besides their functions in lipid absorption, bile acids/bile alcohols are regulatory molecules for a number of metabolic processes. Their effects are structure-dependent, and numerous metabolic conversions result in a complex mixture of biologically active and inactive forms. Advanced methods are required to characterize and quantify individual bile acids in these mixtures. A combination of such analyses with analyses of the proteome will be required for a better understanding of mechanisms of action and nature of endogenous ligands. Mass spectrometry is the basic detection technique for effl uents from chromatographic columns. Capillary liquid chromatography-mass spectrometry with electrospray ionization provides the highest sensitivity in metabolome analysis. Classical gas chromatography-mass spectrometry is less sensitive but offers extensive structuredependent fragmentation increasing the specifi city in analyses of isobaric isomers of unconjugated bile acids. Depending on the nature of the bile acid/bile alc...

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High-performance liquid chromatographic-electrospray mass spectrometric analysis of bile acids in biological fluids

Cited by 64 publications

References 27 publications

Taurohyodeoxycholic acid protects against taurochenodeoxycholic acid-induced cholestasis in the rat

Taurohyodeoxycholic acid protects against taurochenodeoxycholic acid-induced cholestasis in the rat

Key discoveries in bile acid chemistry and biology and their clinical applications: history of the last eight decades

Bile acids: analysis in biological fluids and tissues

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