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Review: Bile Acid Analysis

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 Review article Int J Pharm Biomed Sci 2012, 3(2), 28-34
ISSN No: 0976-5263

Research Drops
PharmaInterScience Publishers

Review on bile acid analysis

Kirti Rani Sharma*
Amity Institute of Biotechnology, J3- block,
Amity University Uttar Pradesh, Noida,
Sec-125, Gautam Buddha Nagar, Noida-
201303 (UP), India.
*Correspondence:
Dr. Kirti Rani Sharma
Tel: +91 120 4392946
E-mail: krsharma@amity.edu,
kirtisharma2k@rediffmail.com
Bile acids are made in the liver by the cytochrome P450-mediated oxidation of
cholesterol. They are conjugated with taurine or the amino acid glycine, or with a
sulfate or a glucuronide, and are then stored in the gallbladder, which concentrates
the salts by removing the water. Bile acids serve other functions, including
eliminating cholesterol from the body, driving the flow of bile to eliminate
catabolites from the liver, emulsifying lipids and fat-soluble vitamins in the
intestine to form micelles that can be transported via the lacteal system, and aiding
in the reduction of the bacteria flora found in the small intestine and biliary tract.
Conjugated bile acids are more efficient at emulsifying fats because, at intestinal
pH, they are more ionized than unconjugated bile acids. Several assays have been
used to determine both total and individual bile acids in biological fluids. The
methods that have been used specifically to analyze serum TBA (total Bile Acid)
are gas-liquid chromatography (GLC), High Performance Liquid Chromatography
(HPLC), enzymatic assays and enzyme cycling assays. GLC and HPLC methods
are not commonly used in clinical laboratories where automated clinical chemistry
analyzers are used for most of chemistry tests including TBA testing. The
enzymatic assay is now mainly used in small laboratories where TBA test are in
lyophilized powder form and manual reconstitution steps are needed before use. At
present, the most widely used TBA test in clinical laboratories is the enzyme
cycling method. That is a liquid-stable assay and ready to use for all types of
automated chemistry analyzers.
Key words: Cholic acid, Chenodeoxycholic acid, Deoxycholic acid, Lithicholic
acid, Hepatobiliary diseases

Received: 17 Apr 2012 / Revised: 27 Apr 2012 / Accepted: 29 Apr 2012 / Online publication: 25 May 2012

1. INTRODUCTION
Bile acids are C24 steroids with a hydroxyl-group at 3-α
position which are biosynthetically derived from total bile
Acid cholesterol in the liver. The primary bile acids, cholic
acid and chenodeoxycholic acid are formed in the liver and
conjugated with glycine and taurine before passing to gall
bladder. Through the action of intestinal bacteria, the primary
bile acids may be dehydroxylated to secondary bile acids,
principally deoxycholic acid and lithicholic acid [81]. Thus,
bile acids are formed from cholesterol in the liver, secreted in
the bile, reabsorded from the intestine and re-circulated back
to the liver through an enterohepatic pathway. Synthesis of
bile acids is a major route of cholesterol metabolism in most
species other than humans. The body produces about 800 mg
of cholesterol per day and about half of that is used for bile
acid synthesis. In total about 20-30g of bile acids are secreted
into the intestine daily. About 90% of excreted bile acids are
reabsorbed by active transport in the ileum and recycled in
what is referred to as the enterohepatic circulation, which
moves the bile salts from the intestinal system back to the
liver and the gallbladder. This allows a low rate of daily
synthesis, but high secretion to the digestive system. Bile is
also used to break down fat globules into tiny droplets. Bile
acids play important role in the emulsification of dietary fat,
activation of lipases and absorption of lipids through
intestinal mucosa [28]. Serum bile acids have importance in
the diagnosis of hepatobiliary diseases [55]. Determination of
total bile acids in serum has advantage in the diagnosis of
adrenocortical and hepatobiliary diseases [6]. In the adult
man, the total bile acid pool is 1 to 3g which recycles
approximately ten times per day in enterohepatic circulation
[88]. Thus serum bile acid determination provides
information in certain clinical situation that is not affordable
by routine tests of liver function [4]. Bile acid malabsoption
was found in inflammatory bowel diseases as well as the
incidence of gall stones is elevated by about twofold in
crohn’s diseases [41]. The concentration of bile acids was
significantly increased by high fat feeding and intestinal

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Kirti Rani Sharma, Int J Pharm Biomed Sci 2012, 3(2), 28-34
permeability was increased in jejunum only but not in the
duodenum/ileum [78].
1.1. Metabolism of bile acids
The conversion of cholesterol into bile acids is
quantitatively the most important mechanism for degradation
of cholesterol. In a normal human adult approximately 0.5g
of cholesterol is converted to bile acid per day [95]. The
conversion of cholesterol into primary bile acids (cholate and
chenodeoxycholate) in liver with the help of 7-α hydroxylase
is the rate limiting step [90]. Thus, the reduction in bile acids
caused by impaired 7-α hydroxylation rather than 12-α
hydroxylation that lead to defective feedback control
regulation bile acid synthesis in liver diseases particularly in
liver cirrohosis [68, 88, 89, 19]. The bile salts such as
glycholic acid and taurocholic acid are amphipathic
compounds which are act as biological detergents those
coverting dietary fats into mixed micelles of bile acid and tri-
glycerols [80].
1.2. Bile acid in pathological conditions
Bile acid determination is important to study bile acid
profile to know the exact type of metabolic abnormality
present in the body. The glycine/taurine was increased in
patients with ileal diseases or resection [18, 1, 42]. Biological
half lives of bile acids were prolonged and synthetic rates
were markedly reduced in patients with cholestasis [49]. The
cholic acid synthesis was more reduced than
chenodeoxycholic acid synthesis due to decreased activity of
12-α hydroxlyase in parenchymal liver diseases [88]. The
propostion of cholic acid had been more in liver than large
duct obstruction in cholestasis [23]. The serum bile acid level
was increased as result of decreased hepatic extraction of bile
acids from portal blood with increased spillover into systemic
circulation in hepatocellular diseases [9, 40]. In acute and
chronic cholestatic disorders, cholic acid conjugates had been
predominated [23]. Taurine level had been measured in
hepatic bile acid in liver biopsies and observed the hepatic
accumulation of taurine in biliary obstruction [29]. The
synthetic rate, pool size and fractional turnover of cholic acid
were dramatically reduced but concentration of
chenodeoxycholic acid was remaining unchanged in cirrhosis
[17]. An elevated level of serum bile acid had been proposed
as a highly specific and moderately sensitive sign of liver
diseases [42]. Mostly taurine and glycine conjugated bile
salts were significantly increased in cirrhosis [50]. Serum bile
acid was increased in either total or individual concentrations
in infants with cholestatic disease [85]. Elevated postprandial
cholyglycine (CG) level (48%) was more sensitive indicator
of pruritus during pregnancy [44]. About of 10-100 fold
increased level of total and individual conjugated bile acids
were found in cirrohotic patients and hepatocellular diseases
[22].
©2012 PharmaInterScience Publishers. All rights reserved.
29
2. DETERMINATION OF SERUM BILE ACID
Abnormal bile acid metabolism is often found in patients
with adrenocortical and hepatobiliary diseases, where total 3-
α hydoxrysteroid in urine and total bile acids in serum are
determined [6,13]. Many methods are available for
determination of bile acids in serum, bile and faces of
individuals. A variety of methods have been reported such as
chemical [9], thin layer chromatography [14], high
performance liquid chromatography [37,52],
radioimmunoassay [94], enzyme linked colorometric and
radioimmunoassay [26], mass spectrometry [72], tandem
mass spectrometry [24], gas chromatogaraphy using high
resolution glass capillary columns and mass spectrometry
[69], gas chromatography [16], gal liquid chromatography
[87,3], luminometric method [81], UV method for bile
[75,76], enzymatic colorimetric method [10] and enzymatic
fluorimetric method [48,28].
2.1. Chemical method
Earlier, chemical method was used for determination of
bile acid which was colorimetric and gives quantitative ratio
of trihydroxy-dihydorxy bile acid in serum of patients of liver
and biliary tract diseases [9].
2.2. Chromatographic methods
2.2.1 High performance liquid chromatographic method
(HPLC)
An accurate and sensitive high performance liquid
chromatographic method was developed for assay of free as
well as glycine and taurine conjugated bile acids in serum by
using post-column reaction after group separation of serum
bile acid fractions by ion-exchange chromatography on
piperidinohydroxy-propyl Sephadex LH-20 [51]. The method
for separation and determination of bile acids was developed
by HPLC using immobilized 3-α hydroxysteroid
dehydrogenase [37]. HPLC method was used for extraction
of bile acids from serum by reverse-phase liquid
chromatographic process with an aftadecylsionae column
[42, 30]. HPLC method was used with fluorescence detection
and used dansyl hydrazine as pre-labeling reagent for
determination of bile acids and their conjugates [38]. Micro
HPLC method was described with an immobilized 3-α
hydroxysteroid dehydrogenase of free and conjugated bile
acid in human sera [35]. An enzymatic method was used with
HPLC for the estimation of serum conjugated and
unconjuagted bile acids in young infants with intrahepatic
cholestasis (idiopathic neonatal hepatitis) or extrahepatic
cholestasis (preoperative extrahepatic biliary atresia) [85]. A
method for quantification of individual serum bile acids in
patients with liver diseases by HPLC which was reformed by
using a 5 micron C-18 reversed phase packed in a 300×4 mm
(internal diameter) column [50]. HPLC method was
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Kirti Rani Sharma, Int J Pharm Biomed Sci 2012, 3(2), 28-34
developed for estimation of free and conjugated bile acids in
human faeces [15]. A new high performance thin-layers
chromatography and denstometry method was also developed
for analysis of individual bile acid and their glycine/taurine
conjugates in duodenal juice [61]. An accurate and sensitive
HPLC method with immobillzed 3-α hydroxysteroid
dehydrogenase column was developed for direct
measurement of urinary bile acids of noncholestatic and
cholestatic infants [84,92]. A new improved HPLC method
using paried-ion chromatography was developed for
measurement of conjugated bile acids in serum [32]. New
advanced HPLC method was also developed with microbe
column for analysis of bile acids and sterols [54]. The
determaination of conjugated bile acids in human serum by
using post column enzyme reaction and off-line fluorimetric
determination [82] as well as in human bile [63] and gastric
juices [21] were developed. A simple reverse phase high
performance liquid chromatography with evaporative light
scattering of mass detection method was developed for
simultaneous analysis of free, glycine and taurine-amindated
bile acids [62]. A mixed HPLC method with
chemiluminescence detection was made for estimation of bile
acids in human urine [34]. A simple HPLC method was used
for quantitative estimation of 10 3-α hydroxyl bile acids in
pediatric serum samples [20]. A unique HPLC method was
developed for quantifying taurine conjugates of bile acids in
serum which involved firstly, the removal of free amino acids
via soild phase extraction of serum; secondly, the reaction of
extracted serum with enzyme choloylalycine hydrolase,
which liberated the taurine from conjugated bile acids and
third step was the reversed phase HPLC separation of O-
phthalicdiccarboxaldehyde [52].
2.2.2. Gas liquid chromatography (GLC)
Gas liquid chromatographic methods were developed for
analysis of individual bile acids in serum and bile [87] as
well as serum bile acid level in patients of primary biliary
cirrhosis [4]. A modified GLC method was developed for
determination of serum bile acids in diagnosis of
hepatobiliary diseases and with radioimmunoassay [55]. GLC
method along with the use of 3-α steroid dehydrogenase was
used for measurement of conjugated and unconjugated bile
acid concentrations [73]. Rapid and sensitive GLC method
was described for bile acid analysis by using
hexafluoroisopropanol plus trifluoroacetic anhydride for the
derivatization of bile acids [5]. A modified GLC method was
developed for estimation of fasting and postprandial serum
bile acid level in which separation of bile acids was done by
ion exchange chromatography with the combination of Helix
Pomatia and cholyglycine hydrolase followed by GLC/ mass
spectrometry for release of analyzed bile acids [31]. GLC
method was used for measurement of qualitative and
quantitative profiles of conjugated bile acids in serum in ileal
resection and bacterial overgrowth and total serum
unconjugated bile acid level [70]. A multicomponent analysis
©2012 PharmaInterScience Publishers. All rights reserved.
30
of bile acid in faeces was done by anion exchange and
capillary GLC [39]. GLC-mass spectrometry method was
also used for characterization of serum and urinary bile acids
in patients of primary biliary cirrhosis [3].
2.2.3. Gas chromatography (GC)
GC methods with high resolution glass capillary columns
and mass spectrometry were used for analysis of bile acids in
serum of patients of liver diseases/ileal resection [69] and in
patients of cystic fibrosis [70]. An advanced capillary GC
method/negative ion chemical ionization mass spectrometry
was used for quantification of bile acids [47]. GC method
was also used for measurement of bile acis in liver in end
stage chronic cholestatic liver diseases [16].
2.2.4. Other advanced chromatographic methods
Quantitative determination of bile acids and their
conjugates was done by thin layer chromatography and
purified 3-α hydroxysteroid dehydrogenase [14]. The
determination of free bile acids was done in pharmaceutical
preparation by packed column supercrtitical fluid
chromatography [65]. Ion spray liquid chromatographic/mass
spectrometric method was used for characterization of bile
acid and their conjugates [91]. Bile acid analysis was also
done in various biological samples by ultra performance
liquid chromatography with electrospray ionization- mass
spectrometry [27] and by liquid chromatography with
electrospray ionization mass spectrometry in fecal materials
too [8].
2.3. Mass spectrometry (MS)
A highly sensitive MS method was done for estimation of
bile acids with lower detection limit of 0.01-0.05 nmol [72].
MS method was also used for identification of biliary bile
acids in the bile of gall stones patients before and during
treatment with chenodeoxycholic acid [77]. Liquid
chromatography electrospray tandem mass spectrometric
determination of bile acids was done in biological fluids [56].
New advanced GS methods with matrix-assisted laser
desorption/ionization was done for quantification of bile
acids in urine samples [46]. A tandem mass spectrometry was
also used in the study of fatty acids, bile acids and steroids
with a combination of sensitive gas chromatography and
selective electron ionization mass spectrometry [24].
2.4. Radioimmunoassay
Radioimmunoassay was introduced for determination of
conjugated cholic acid and sulfoglycolithocholic acid in
serum and bile [94]. Radioimmunoassay was also used for
determination of bile acid in serum in the diagnosis of vinyl
chloride hepatotoxicity [43]. The measurement of primary
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Kirti Rani Sharma, Int J Pharm Biomed Sci 2012, 3(2), 28-34
bile acids was done in serum of children suffering from celiac
disease by radioimmunoassay [12].
2.5. Bioluminescence
Bioluminescence assay was done for estimation of bile
acids using a co-immobilized 3-α hyroxysteroid
dehydrogenase, diaphorase and bacterial luciferase [67]. A
simple bioluminescence assay was done for detection of bile
acid along with the serum glutamate pyruvate transaminase
determination in the serum of patients of hepatic injury [66].
2.6 Various methods
Continuous-flow analysis of 3-α hydroxysteroid was done
by using immobilized 3-α hydroxysteroid dehydrogenase [6].
Nuclear magnetic resonance spectroscopy of bile acids was
also done [93]. A new potentiometric method was developed
to study the equilibria in aqueous solution for system copper
(II)/ bile acid/peptide which was specific to access co-
operative binding between small peptide and bile acids in
presence of copper (II). An isotope dilution technique was
also used for study of bile acid kinetics and biliary lipid
compostion in cystic fibrosis [79]. Proton magnetic resonance
assay was described for estimation of total and taurine-
conjugated bile acids [35]. Simultaneous determination of
free and conjugated bile acids in serum was done by
cyclodextrin-modified micellar electrokinetic
chromatography [86].
2.7. Method using free enzymes
Enzymatic methods are new and universally used for the
bile acid determination. These are relatively specific, rapid
and easy to use. Enzymatic fluorometric micro assay was
developed for measurement of ratio of primary to total bile
acids in serum for clinical significance in patients of liver
diseases [33]. A comparison was made in between enzymatic
fluorescence and gas chromatography by a linear regression
plot which revealed that correlation was fair of r= 0.78 [63].
2.7.1. Enzymatic-colorimetric method
An enzymatic-colorimetric determination of total bile
acids was done in gastric juices [10]. Enzymatic colorimetric
assay was done in serum of 3-α hydroxyl bile acids
concentration without extraction [58, 83]. Centrifugal
analyzer with spectromphotometric method was developed
for enzymatic measurement of total bile acids [28]. An
accurate enzymatic measurement of faecal bile acid in
patients with malabsorption was described which helped to
elucidate the cause of diarrhea and steatorrhea [57].
©2012 PharmaInterScience Publishers. All rights reserved.
31
2.7.2. Enzymatic fluorimetric method
An enzymatic –fluorimetric method was used for
estimation of serum total bile acids in jaundice patients [48],
with distinct diagnostic value of serum bile acid in liver
disease [2], during fasting serum of patients of liver diseases
[73] and in patients of cirrhosis [36]. A new modified
enzymatic fluorimetric method using a highly purified 3-α
hydroxysteroid dehydrogenase was used to determine fasting
bile acid concentration in patients with liver diseases [13].
Simple and sensitive enzymatic flurometric methods were
described for estimation of serum total 3-α hydroxy bile acids
[45, 7, 33]. Advanced enzymatic-fluorimetric measurement
of primary bile acids was described in serum samples with
IL-Multistate III fluorescence Light-Scattering centrifuged
analyzer [53]. Enzymatic method was used for analysis of
bile acid by using dual-beam spectrophotofluorimetry [71].
2.7.3. Methods using immobilized enzymes
Continuous flow analysis was described for estimation of
bile acids by using immobilized 3-α hydroxysteroid
dehydrogenase and diphorase individually onto cellulose gall
beads [6]. HPLC method by using immobilized 3-α
hydroxysteroid dehydrogenase was used for estimation of
bile acids [51], with efficient immobilized column in alkaline
medium, triammonium phosphate buffer pH 11 [30], with
electrochemical detector [37] and fluorometric method too
[38]. A bioluminescence method was introduced for assay of
3-α hydroxyl bile acids in serum using co-immobilized 3-
αsterid dehydrogenase, diaphorase and bacterial luciferase
onto Sepharose 4B through CNBr linkage [66, 67]. Modified
enzymatic methods were described for discrete analysis of
bile acid was by using immobilized 3-α hydroxysteroid
dehydrogenase and diaphorase individually onto arylamine
glass beads through diazotization [59] and alkyalmine glass
beads through glutaraldehyde [60].
3. COMMERCIAL AVAILABILITY OF METHODS OF
SERUM BILE ACID DETERMINATION
Based on enzymatic colorimetric method, sigma bile acid
kit supplied by Sigma Aldrich, USA and enzymatic-
fluormetirc method, enable kit is by Nyeggard and co., A/S,
Oslo, Norway [45] and sterognost 3-α automated kit
9Nyegaard an d Co. A/S, Oslo, Norway [14].
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 Kirti Rani Sharma, Int J Pharm Biomed Sci 2012, 3(2), 28-34
4. CONCLUSION
The efficient hepatic clearance of bile acids from portal
blood is responsible for serum concentration upto 10μmol/L
in normal person. An elevated fasting level of bile acid is
found only in impaired hepatic clearance which is a sensitive
indicator of liver disease including cirrhosis, hepatitis,
cholestasis, cholangitis, Wilson's disease, acute hepatitis,
chronic hepatitis and liver sclerosis. Fasting serum bile acids
is helpful in the diagnosis and prognosis of liver disease.
Abnormal levels in fasting patients or immediately after a
meal is good indicator for liver disease and damage, impaired
liver function, intestinal dysfunction and gall bladder
blockage due to gall stone formation. Thus, bile acid
measurement may detect some forms of liver disease earlier
than standard liver tests because bile acids levels correspond
to liver function, rather than liver damage.
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