CLIN. CHEM. 27/10. 1698-1703 (1981)
Specific 1251-Radioimmunoassay for Cholyiglycine, a Bile Acid, in Serum
Phillip Miller, Stephen Weiss, Martha Cornell, and Joan Dockery
The concentration of the conjugated bile acid, cholyiglycine, in serum is a sensitive and specific indicator of hepatic function. We describe a convenient, specific, and
precise radloimmunoassay for cholylglycine, in which
125l-labeled cholyiglycyltyrosine is used as tracer. In addition, a blocking agent in the buffer system eliminates
binding of bile acids to serum albumin. Therefore no extraction is required. We found no interference by (a) abnormal concentrations of albumin or gamma-globulin, (b)
lipemicsera,(C) hemolyzed sera, (c anticoagulants,
or (a)
various commonly used drugs. The reference interval for
fasting subjects is estimated to be 0.0 to 0.6 mg/L. Our
clinical studies show that serum cholylglycine concentrations are usually abnormal in most hepatobiliary diseases, such as viral hepatitis, alcoholic liver disease,
cirrhosis, and pediatric liver diseases.
AdditIonalKeyphrases: liver disease
pediatric chemistry
.
reference interval
Bileacids,synthesized
and conjugated under the influence
of hepatic enzymes (1), are almost completely confined to the
enterohepatic circulation by intestinal and hepatic transport
systems (2). Approximately 95% of the bile acids secreted into
the intestine in bile are re-absorbed from the intestine into
the portal blood (3). In the absence of hepatobiliary disease,
almost all of the bile acids are cleared from portal blood by
hepatic uptake; the remainder spills into the peripheral circulation (4).
Many reports suggest that the bile acid concentration in
serum, whether measured during fasting or postprandially,
is a sensitive and specific indicator of hepatobiliary dysfunction (5-10). Although most of this work has been based upon
measurement of total bile acids, several reports demonstrate
the clinical value of measuring individual bile acids, particularly conjugates of cholic acid (11-14).
The clinical utility of CG’ measurement arises from the fact
that in health the liver efficiently extracts it from the portal
blood, 80% to 90% of it being removed in a single pass (15).
When hepatic function is altered, more CG is spilled into the
peripheral
circulation,
increasing its concentration
in
serum.
In addition, there is an active-transport
in the distal
(15).
ileum,
which
returns
mechanism for CG
it to the portal
blood
Currently, three techniques are used to estimate bile acids
in serum: gas-liquid
chromatography
(17,
18),
an enzymic
fluorometric
method (19,20), and radioimmunoassay
(21-23).
Considering
the sensitivity
of the radioimmunoassay
technique and the potential clinical significance of CG, we developed such an assay for CG, which features an iodinated tracer
Diagnostics
Division, Abbott Laboratories, Abbott Park, North
Chicago, IL 60064.
Nonstandard abbreviations
used:CG, cholyiglycine; BIT, ratio
of bound to total counts per minute;B/B0, ratio of bound to zerostandard bound countsper minute.
Received April 6, 1981;accepted
1698
CLINICAL
CHEMISTRY,
June
19, 1981
Vol. 27, No. 10, 1981
and a buffer system that eliminates the need for ethanol extraction.
Materials and Methods
Reagents
The following reagents were obtained from commercial
Cholylglycine (Sigma Chemical Co., St. Louis, MO
sources.
63178), bovine serum albumin (Sigma), ethylcarbodiimide
hydrochloride (Story Chemical Co., Muskegon, MI 49444),
[3Hjcholylglycine (New England Nuclear, Boston, MA 02118),
8-anilinonapthalene1-sulfonic acid (Eastman
Kodak,
Rochester, NY 14650), bovine gamma-globulin (Miles Research Products, Elkhart, IN 46515), polyethylene glycol of
average molecular mass 6000 (Union Carbide Corp., NY
10017),barbitaland sodium barbital (Gaines Chemical Co.,
Pennsville,NJ 08070), pulverized charcoal RC (Pittsburgh
Activated Carbon, Pittsburgh, PA 15222), normal human
serum (Interstate
Blood Bank, Memphis, TN 36118), thi-
merosal (Eli Lilly, Indianapolis, IN 46206), absolute ethanol
(U.S. Industrial Chemicals Co., New York, NY 10016), and
Freund’s
complete and incomplete adjuvants (Difco Laboratories, Detroit, MI 48232).
Procedures
Tracer.
Cholylglycyltyrosine
was prepared by coupling
cholic acid to glycyltyrosine.
This synthesis involved preparation of two stable intermediates,
cholic acid hydrazide and
butoxycarbonylglycyl-1-tyrosine
benzyl
ester. The cholylglycyltyrosine was then characterized by structural analysis
and for purity by thin-layer chromatography (isopropanol/
H20/pyridine, 16/311 by vol), infrared spectroscopy-nuclear
magnetic resonance spectroscopy, and elemental analysis. The
cholylglycyltyrosine
was iodinated by the Chloramine T
method described by Hunter and Greenwood (24). The iodinated product was purified on a column of LH-20 Sephadex,
the effluent of which was monitored by thin-layer chromatography. Its purity exceeded 98%, with a maximum binding
of 85% with use of excess antibody. Specific activity, as estimated from the degree of incorporation, was 186 kCi/mol.
Antigen
and antibody.
The CG used to prepare
the immunogen was >99% pure, as judged by thin-layer chromatography, potentiometric titration, elemental analysis, nuclear
magnetic resonance, and mass spectroscopy. We followed the
method of Simmonds et al. (21) in preparing the immunogen
by coupling CG to bovine serum albumin with use of watersoluble ethylcarboduimide. Tritiated CG (3HCG) was added
to the reaction and the coupling efficiency (percent of 3HCG
bound to bovine serum albumin) was determined. After dialysis, the immunogen was further purified from noncovalently bound CG by elution through a column of G-25
Sephadex. This material was then used to immunize and
harvest antisera from New Zealand White rabbits similar to
that described by Simmonds et al.(21). Animals were injected
at multiple sites with 100 jig of the antigen emulsified in 1.0
mL of Freund’s complete adjuvant. This procedure was repeated for four consecutive weeks, followed by booster injec-
Table 1. Cross Reactivity with Various Bile Acids
and Non-Bile-Acid Steroidal Compounds
50
40
Mean cross
compounda
Glycocholic acid
Cholic acid
Taurocholic acid
Deoxychollc acid
Glycodeoxychollc acid
Taurodeoxycholic acid
reactIvIty,
-t
I-.
14.5
3.2
0.1
0.4
0.3
Sulfochenodeoxycholic acid
Estradlol
Prednisone
Testosterone
Digoxin
Digitoxin
Cortisol
Progesterone
Cortisol acetate
Cholesterol
The fist 12 conWo.ruds listed were assayed
atconcentrations
of0.01,0.10,
1.0. and 10.0 mg/L; the last 13 compounds, assayed at four concentrations
ranging between 0.001 and 30.0 mg/L, showed no cross reactIvity.
5The percentage cross reactivity Is the mean value f the fow concentratIons
listed.
tionsof 100 jig of antigen in Freund’s incomplete adjuvant at
monthly intervals.
Buffer. The buffer (pH 6.8)isa 60 mmol/L solutionof diethyl barbital containing 7.5 g of bovine gamma globulinand
1.2g of 8-anilino-1-naphthalenesulfonicacid.
Standards. Standards were prepared from a stock solution
(CG in water/ethanol, 10/1 by vol) by dilutionto produce
concentrations of 0.25,2.50,10,and 40 mg/L. A set of stan-
dards was prepared in a protein-containing
buffer solution
globulin, 50 mmol of phosphate, and 9 g of sodium chloride); a second set was prepared
in bile-acid-free
human sera (21).
Radioimrnunoassay
procedure.
We used two procedures.
The first, based on the method of Murphy et al. (25) involving
a tritiated-CG tracerand ethanol extraction,was used as a
reference assay.In our proposed method (Figure 1) we use
‘251-CG tracer without extraction,because the barbital!
anilinonaphthalenesulfonicacid buffereffectively
blocks the
1 g of bovine
10
0.25
1.0
gamma
2.5
3.0
10.0 40.0
CG mg/I
3.3
acid
Glycolithocholic acid
Taurolithocholic acid
Sulfolithocholic acid
Unconjugated
(per liter,
20
2.8
1.7
Taurochenodeoxycholic acid
Lithocholic acid
a
30
1.0
Chenodeoxycholic acid
Glycochenodeoxycholic
% b
100
7.8
16.5
Fig. 1. Typical standard curve for CG RIA
to protein of an anion such as CG (26). The assay
procedure requires pipetting 25 jiL of standards or unknowns,
200 jiL of tracer, and 200 jiL of antisera into 12 X 75mm test
tubes; for nonspecific binding tubes, 200 jiL of buffer is added
binding
inplace of the antisera.Assay tubes are then incubated for 1
h at room temperature, followed by 2.0 mL of polyethylene
glycol.Tubes are then centrifuged (1000 X g, 10 mm), the
supernates
decanted, and the radioactivity
remaining in each
tube is determined by counting in an Autologic gamma
counter (Abbott Laboratories, North Chicago, IL 60064).
Assay results were then calculated
based on the log-logit
transformation
(27).
Antigen and Antisera Characteristics
The
antigen,
characterized
based
on the
percentage
of
3HCG incorporated into the purified conjugate, was found to
have 12 mol of CG per mole of bovine serum albumin.
Antisera,harvestedafter three months, gave a typicaltiter
of 1:1000 based on 50% binding of 1 pmol of tracerper tube.
Antibody was characterizedfor affinityand capacity by use
of a Scatchard plot. The affinitywas 6.1 X 106 L/mol, with a
binding capacityof2.1 X 10 molfL for the 1251tracerforthe
anilinonaphthalenesulfonicacid buffer system. Antibody
specificity was also evaluated(Table 1)by assayingfour widely
differingconcentrationsof compounds with structuressimilar
to CG. The mean crossreactivity
of these fourconcentrations
was determined. This was done in thisway because many of
the compounds have limitedsolubilityand low crossreactivity,and concentrationshigh enough to cause 50% displacement of tracer are not attainable.
Assay Characteristics
A typical standard
curve prepared
with
per tube (see Figure 1) has a B0 and
nonspecific
binding of approximately
60% and 5% BIT, respectively, and a 50% intercept of about 2.2 mg/L with a least
detectable dose of 0.8mg/L (0.02jimol/L).
Log-logitlinear
Standard
curve.
use of 1 pmol of tracer
Table 2. Precision Data for the Present Radioimmunoassay of Cholylglycine
Run 1
Run 2
Control Pool 1
mg/L
0.306
0.287
CV, %
7.8
6.1
0.839
7.0
0.802
3.6
0.88 1
3.9
0.923
4.8
0.863
6.3
0.857
7.3
2.34
2.7
2.38
2.5
2.43
3.8
2.35
3.8
2.38
1.7
2.37
3.3
S.rum
Run 3
Run 4
0.314
0.390
10.1
4.3
Int.rassay
a
0.324
14.0
Grand b
0.321
13.4
Control Pool 2
mg/L
CV, %
Control Pool 3
mg/L
CV, %
#{149}
Measwe of the variation of the means
ofthe
Intra-assays. 5Varlatlon of the 25 independent observations about the grand mean.
CLINICALCHEMISTRY,Vol. 27, No. 10, 1981 1699
KINETICS (20- 30#{176}C)
Table 3. Analytical Recovery of Cholylglycine
Added to Eight Normal Human Sera
-‘Omg/ICO
#{149}
S
60
Concn found
after adding
CO 10.4 mg/L
InItIal CO
70
Recovery,
%
0.25 mg/L CO
50
mg/L
0.20
11.29
98.6
105.0
112.0
107.0
0.35
10.70
99.5
0.28
10.54
11.21
0.33
0.32
11.83
11.53
11.73
11.83
0.15
0.16
0.10
40
Z
20
-#{149}--
10
109.0
11.0
113.0
x=
-
30
-.2.5
-
-
..-.
1.0 mg/L CO
mg/L
10 mg/I
l4Omg/LCO
-&
-
0
ii
4
6
CO
CO
rI,J,,Srg,_ur,
22
TiME(H
107.0
Fig. 2. Effect on percent binding of incubation time at room
temperature (20 to 30 #{176}C)
from 0 to 22 h
regression
of the standard curve data gives a correlation
coefficient
exceeding
-0.995, with a slope of -0.9 to -1.0.
Precision.
We evaluated the reproducibility
of the assay by
assaying three serum pools in replicates of five or 10 over four
separate runs on four different days,using one lot of material.
Interassay, intra-assay, and total variability (the grand mean
Ntsthers at riit are millIgrams of CO per lIter. KInetic study shows no change
In percent bouid vs time for IndIcated concentrations of CO at room temperatwe
when Incubated longer than 1 h
developed by Rodbard and Lewald
(27),
is 80 igfL
(0.2 mol!
L).
Parallelism.
Interference
was evaluated by parallelism
over the four runs) were computed (Table 2).
studies.
Sera
containing
abnormal
concentrations of CG were
Accuracy.
Accuracy of the assay was judged from the anadilutedwith a solution containing 40 g of human serum allytical recovery of CG added to normal human sera and pabumin and lOg of bovine ganima.globulin per liter of isotonic
tients’ sera that contained above- or below-normal concensaline (9 g of NaCI per liter). Parallelism was seen between the
trations of gamma-globulin. The mean recoveries were 107%,
standard curve and results for the diluted patients’ sera, in100%, and 93%, respectively (Tables 3 and 4).
dicating equivalent reactivity of CG (Figure 3). Additional
Kinetics.
The kinetics of the assay demonstrate
that the
dilution studies with use of the same human serum albumin
percentage bound is essentially constant for all concentrations
after a 1-h incubation
at room temperature
(20 to 30 #{176}C) and gamma-globulin solution described above showed that
lipemic sera (Figure 3), hemolyzed sera,and anticoagulants
(Figure 2).
such as heparmn, tetrasodium ethylenediaminetetraacetate,
Least detectable
dose. The minimum
concentration of CG
citrate, and oxalate do not interfere. Ten and 100 mg of spedetectable with 95% confidence of difference from the zero
cific drugs were added to pooled serum per liter, in testing for
dose for the assay, as determined with a computer program
interference. Penicillin, caffeine, ascorbic acid, phenobarbital,
and 32 other commonly used drugs caused no significant interference. Also, dilution studies demonstrated that nothing
Table 4. Analytical Recovery of Exogenous
in urine or bile would affect assay results (Figure 4).
Cholyiglycine (CG) from Normal and Patients’
Protein effects. We prepared solutions containing various
Sera with Abnormal Concentrations of Gamma
concentrations of human serum albumin and gamma-globulin,
Globulin
to assess the possibility of interference. These solutions inConcn. found
Sample
InitIal CO
concn.
after addIng
CO 9.25 mg/L
Recovery,
%
99
mgIL
98
Hypo-IgO
A
0.16
9.27
98.5
95
B
0.21
9.58
101.0
90
C
D
0.51
1.33
10.00
10.00
106.0
94.0
80
E
0.23
9.87
104.0
5o
F
G
0.16
0.17
9.35
9.65
99.9
102.0
20-
H
0.19
9.30
98.5
10’
100.0
=
5.
Kyper-lgG
A
0.26
9.36
98.4
B
0.11
0.08
9.10
97.3
8.08
9.25
8.61
9.19
86.5
96.6
92.7
97.4
C
D
E
F
0.31
0.13
0.18
=
1700
CLINICAL CHEMISTRY.
Vol. 27, No. 10, 1981
93.0
I
I
10
40
CO mg/I
Fig. 3. Results of assays of patients’ sera with abnormal CG
lipemic sera at various dilutions
concentrations or
The results show parallelIsm and no Interference. The standard ctave Is represented by 0-0
and Ilpemic sara dIluted 1:2, 1:4, 1:8, 1:16 by U-U.
#{149}-#{149}.SerafrompatientswlthabnormalCOdilutedl:2,1:4.
1:8.and 1:16
are represented by A-&
0-0
90
A
so
30’
A
70
A
60
24
:1
so
A
40
AAA
AA
1$
A
20
12
A
10
6
S
1
10
‘p
Unear response Indicate no Interference. Himw bile (#{149}-#{149},
U-U)
dIluted
1:1000, 1:2000, 1:5000, 1:10000. Human wIne (0-0,
- -)
undIluted
anddlluted 1:2,1:4,1:8, 1:18 Blleandwmnewerebothdllutedwlthasolution
contaInInghuman serum albumIn, 40 mgJL and bovine gamma globulIn, 10
mg/L. In IsotonIc saline. A standard curve (A-A)
Is shown for comparIson
20
30
40
50
60
70
________-CONSTANt
60
‘ __O
SO PIOTEIN
40g/L
H10
so,
g/L I4SA 01 H150
NSA
0 TO 40g/I.
CONSTANT HIgO l.Sg/L
vASYINO HSA 0 TO SOg/L
a
20
vations
Table 6. Concentrations of Cholylglyclne in
Serum of Normal Individuals and Persons wIth
Hepatobillary Disease
PatIents
Normal persons
n
179
Alcoholic liver disease
Cirrhosis
1.00
40
Trltiated-tracer and ethanol-extractIon method (25) (x-axls) and 1251 tracer and
ANS method (y-axls) show hI correlation over a wIde range of concentrations.
A, one observation; B, two observatIons; C, ttwee observatIons; 0, four obser-
Acute viral hepatitis
0.50
30
CO mg/L ETHANOl MSTHOO
Fig. 6. Correlation between results by the ethanol-extractIon
procedureand 125l-CGassay
FIg.4. Results of linear dilution of bile and urine
10
10
40
CO mg/I.
CORRELATIONCOEFFICIENT = 0.99
SLOPE= 0.55
INTERCEPT= 0.56 mg/L
nSO
0
03
A
pa
0
70
A
2.50
Fig. 5. Effect of human serum albumin (HSA) and gammaglobulin (HIgG) on results CG assay
No Interference isseenby normal or above-normal concentratIons of albumin
or gamma-globulIn. The constant HIgO with varying HSA Is represented by
U-U,
the constant HSA with vaylng HlgG by 0-0,
andthestandadc*ive
by #{149}-#{149}
29.0 (33.0)
142
10.7 (12.4)
Extrahepatic obstruction
Pediatric liver disease
68
a
CO .g/L
0.20 (0.19)
56
56
23
a
Mean (and SD), mg/I
11.0
27.99
(7.7)
(40.2)
26.2 (23.0)
Blliary atresIa, intrahepatic cholestasls, or neonatal hepatitis.
tamed,which indicates the validity of the present method
(Figure 6).
Twenty-one
specimens
analyzed
by gas-liquid
chroma-
tography and radioimmunoassay
cluded CG in a concentration of approximately 0.1 mg/L. We
saw no changes in apparent CG concentration with variations
in albumin and gamma-globulin concentrations (Figure 5).
Stability.
Storage of control sera A and B at 2 to 8 #{176}C
for
four days, whether frozen or thawed and refrozen, had no effect on specimen stability (Table 5). Additional studies have
shown CG to be stable in serum at 2 to 8#{176}C.
All components
were freed of bacterial contamination
by sterile filtration
were compared. Linear regressionanalysisof the resultsgave a correlation coefficient
of 0.89 and slope of 1.11 (W. F. Balistreri, personal commu-
nication). Because the gas-liquid chromatographic method
total cholate (free and conjugated) and the radioimmunoassay primarily the conjugate, these data demonstrate
good agreement between results by the two methods.
measures
before study.
Reference Interval
We estimated the fasting reference interval for CG in the
serum to be 0.0 to 0.60 mgfL (0.0to 1.29 imol/L), with a mean
Comparison with Other Methods
Results by the two radioimmunoassay methods correlated
well; a correlation coefficient of 0.99,slope of 0.85,and inter-
were volunteers with no previous history of liver disease,
whose serum gave normal results for liver-status tests, including aspartate
aminotransferase
(EC 2.6.1.1) (28), alanine
cept of 0.05 mg/L for 60 normal-and abnormal sera were ob-
aminotransferase
of 0.196 mgfL (0.42 mol/L)
(Table 6). The “normal”
persons
(EC 2.6.1.2) (28), alkaline phosphatase
(EC
Table 5. StabilIty of CGIn Two Control Sera (A and B) under Various Sample-Storage Conditions
CO found, mg/I
2 to 8 #{176}C
Frozen aIIquots
Thawed
and refroi.n
A
B
A
B
A
B
Day 2
0.46
0.43
5.9
6.0
0.48
0.45
5.8
55
0.48
0.49
6.3
6.4
Day 3
0.40
5.8
0.42
5.7
0.49
6.5
Day4
0.43
5.8
0.41
5.7
0.50
6.0
Day 1
CLINICAL CHEMISTRY, Vol. 27, No. 10, 1981
1701
Table 7. PublIshed RIA Value s for CG or Conjugates of Cholic Acid
Year
Ref. no.
Tracer
ExtractIon
Bile acId
Normal range
Mean (SD)
Concn., mo1/L
1973
3H
none
Conj. cholic
3H
3H
3H
3H
ethanol
dilution
XAD-2
none
Conj.cholic
Conj. cholic
Conj. cholic
CG
1977
1977
21
25
35
14
22
36
37
3H
none
Conj. cholic
125J
1977
32
125
ethanol
none
Conj. cholic
CG
1974
1976
1976
1976
3.1.3.1) (29), total bilirubin
(EC 2.3.2.2) (31).
(30),
and 7-glutamyltransferase
CG in Hepatobiliary
Disease
CG was measured in 142 patients with alcoholic liver disease, 56 patients with acute viral hepatitis, 56 patients with
cirrhosis, 23 patients with extrahepatic obstruction, and 68
patients with pediatric liver disease (Table 6). For more than
98% of these patients, the values exceeded the normal range.
We also assayed serum from 42 patients with diseases not
involving the liver or intestine; almost all the CG concentrations were within the normal range, only two of the 42 patients
having values >0.6 mg/L. No other liver-function test demonstrated this degree of specificity (alkaline phosphatase, nine
abnormal results; bilirubin, five; aspartate aminotransferase,
four; alanine aminotransferase, 10).
Discussion
Previously reported radioimmunoassays give adequate
specificity and sensitivity, but require the use of a tritiated
tracer and also sample pretreatment intended to eliminate
interference by proteins such as albumin, which binds bile
acids (32, 33). Albumin influences the radioimmunoassay
procedure because of its affinity and capacity for each particular bile acid. To eliminate this effect, one either must extract the sample or use a system in which the affinity and capacity of the antibody is much greater than that of albumin.
Alternatively, one can utilize a buffer containing agents that
inhibit albumin binding, thetechnique used withthepresent
assay.
With our assay, analytical recoveries of 93 to 107% are
demonstrated for sera with normal and abnormal protein
concentrations. On extrapolation, results for linear dilutions
of sample pass through the zero intercept. This point is particularly critical because bile acids bind to albumin, and
concentrations of albumin and gamma-globulin can vary
greatly in patients with hepatobiliary disease (34).
Our specificity data show that this assay predominantly
measures the glycine conjugate of cholic acid, which is advantageous because CG is one of the dominant bile acid conjugates in children and adults. This would suggest that CG is
an appropriate
marker of hepatic disease. Additionally,
the
proposed reference interval for adults compares well with
previously
reported values for glycine conjugates or total
conjugates of cholic acid (Table 7). Measurement
of the taurine conjugate is not clinically significant
unless a neonate is
being evaluated, and recent work (38) shows that measurement of primary
bile acids is inappropriate
in neonates
younger than one year. Measurement
of secondary bile acids
is recommended
during the neonatal period (39).
lodinated bile acid radioimmunoassays
that do not require
large sample volume or extraction should be more convenient
for routine use in clinical laboratories.
1702
CLINICALCHEMISTRY,Vol. 27, No. 10, 1981
0.54 (0.04)
0.5-1.8
1.4 (0.3)
0.18-1.25
0.27 (0.03)
0.62 (0.4)
0.51 (0.09)
0.42(0.2)
0.0-1.29
References
1. Danielsson, H., and Sjovall,
J., Bileacidmetabolism.
Ann. Rev.
Biochem. 44, 233-253 (1975).
2. Ponz deLeon, M., Murphy, G. M., and Dowling, R. H., Physiological factors
(1978).
influencing
serum
bile
acid
levels.
Gut
19, 32-39
3.
Dowling, R. H.,Mack,E., and Small, D. M. M.,Effects of controlled
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of theenterohepatic
circulationofbile
salts
by biliary
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A. F.,Dynamics ofthe enterohepatic circulation of bile acids: Postprandial serum concentrations
on conjugates
ofcholicacidinhealth,
cholecystectomised patients and patients with bile acid malabsorption.
N. Engi. J. Med. 291, 689-692 (1974).
5. Sherlock,
S.,and Waishe, V., Blood cholates
innormal subjectsand
inliver disease. Clin. Sci. 6, 223-234 (1948).
6. Bloomer,
J.R.,Allen,
R. M.,andKlatskin,
G.,Serum bileacids in
primary biliary
cirrhosis.
Arch. Intern. Med. 136,57-61 (1976).
7. Kaplowitz,N.,Kok, E.,and Javitt,
N. B.,Postprandial
serum bile
acidforthedetectionof hepatobiliary diseases. J. Am. Med. Assoc.
225,292-293 (1973).
8.Fausa,
0., and Gjone, E., Serum bile
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CLINICAL CHEMISTRY,
Vol.
27,
No. 10, 1981
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