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Radioimmunoassay of serum conjugated cholic acid
39
Clinica Chimica Acta, 72 (1976) 39-48 @ Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
CCA 7894
RADIO~UNOASSAY
SIEGFRIED
OF SERUM CONJUGATED CHOLIC ACID
MATERN *, RUTH KRIEGER
and WOLFGANG GEROK
Medizinische Kfinik der Universitiit Freiburg i.Br. (G. F. R.) (Received February 18th, 1976)
Summary A radioimmunoassay for serum conjugated cholic acid is described using antiserum obtained five weeks after immunization of rabbits with cholic acidbovine serum albumin conjugate. Prior to the radioimmunoassay, extraction of bile acids was performed with Amberlite XAD-2. The displacement curve of glyco[ 3H]cholic acid was linear on a log&log plot from 5 to 80 pmol of unlabelled glycocholic acid. Values for 8 normal fasting subjects ranged from 0.18 to 1.25 E.tmol/l. In 31 fasting subjects with or without liver disease serum values of conjugated cholic acid are related to serum bilirubin. Endoscopie retrograde cholangiopancreatography may influence serum levels of conjugated cholic acid.
Introduction Methods for determination of individual bile acids in serum are usually based on gas-liquid or thin-layer chromatography [l-5]. However, gas-liquid chromatography is time consuming and thin-layer chromato~aphy insensitive. Only recently a sensitive radioimmunoassay for determination of serum conjugated bile acids was first developed by Simmonds et al. [6] and by Murphy et al. [ 71, about ten years after the first report of production of antiserum against cholanic arid-~bumin conjugates 181. This paper describes a radioimmuno~~y, of serum conjugated cholic acid with an antiserum obtained after immunization of rabbits with cholic acid-bovine serum albumin conjugate. Materials and methods Unlabelled bile acids were obtained from Steraloids Ltd. [2,43H]Cholic acid with a specific activity of 0.25 mCi/0.03’7 mg, glycof 3H]cholie acid with a spe* Address correspondence to: Dr. Siegfried Matern, Ned&in&he 56. D-78 Freiburg i.Br., G.F.R.
UniversitWkJinik,
Hugstetter Str.
40
cific activity of 0.25 mCi/0.039 mg and tauro[3H]cholic acid with a specific activity of 0.25 mCi/0.038 mg were obtained from New England Nuclear, Boston, Mass. The scintillation liquid was Unisolve 1 obtained from Koch-Light Ltd., England. Liquid scintillation counting was done in a Packard Tricarb scintillations spectrometer, model 3380. Complete Freund’s adjuvant was supplied by the Difco Laboratories. Polyethylene glycol and Amberlite XAD-2 were purchased from Serva (Heidelberg), Sephadex LH-20 from Pharmacia (Uppsala/Sweden), bovine serum albumin from Beringwerke (Marburg) and HiEff 8BP from Applied Science Laboratories, State College, Pennsylvania. All reagents (Merck AG, Darmstadt) were of analytical grade and all solvents (Merck AG, Darmstadt) were redistilled. Preparation of antigen Cholic acid was rendered antigenic by coupling to bovine serum albumin with a mixed anhydride reaction as described by Erlanger et al. [9] and by Murphy et al. [ 71. The cholic acid-bovine serum albumin conjugate contained 12 mol of bile acid per mol albumin determined by gas-liquid chromatography [lo]. Protein was determined by the method of Lowry et al. [ 111. Immunization Antiserum was obtained after 4 weekly injections of 1 mg of cholic acidbovine serum albumin conjugate emulsified with Freund’s complete adjuvant into the dorsal skin in each of 6 female rabbits as described previously [ 12,131. Radioimmunoassay Prior to the radioimmunoassay extraction of bile acids was performed by passing 1 ml of serum through a column of Amberlite XAD-2 as described by Makino and Sjijvall [ 51. The evaporated eluate from Amberlite XAD-2 was reconstituted to a volume of 1 ml with ethanol and diluted with 0.01 M potassium phosphate buffer, pH 7.4 to 5 ml. Aliquots of OJ ml were assayed. The radioimmunoassay was performed as described by Simmonds et al. [6] with the following minor modifications: In each tube of the assay were placed either 8000 cpm glyco [ 3H]cholic acid or 8000 cpm [ 3H]cholic acid and in each tube of the standard curve was ethanol in a dilution of 1 : 50. Standard curves obtained with or without this ethanol dilution were identical. The sample values were extrapolated from a straight line standard curve, which was obtained by plotting logit of (B--N)/(B,--N) X 100 against the logarithm of the dose (B, antibody bound labelled bile acid in cpm; BO, antibody bound labelled bile acid in cpm at zero dose unlabelled glycocholic acid; N, non specific bound labelled bile acid in cpm). The standard curve was linear between 5 and 80 pmol of unlabelled glycocholic acid with glyco [ 3H]cholic acid as well as with [ 3H]cholic acid (example Fig. 1) as labelled bile acid. Gas-liquid chromatography of serum nonsulphated bile acids After extraction of serum bile acids with Amberlite XAD-2 [5] the non-sulphated bile acids separated on Sephadex LH-20 as described by Cronholm et al. [ 141 were hydrolysed and the methyl ester trimethylsilyl ether derivatives [ 151
41
DOS.
glycochollc
rcld
(pmOl@S)
Fig. 1. Standard curve in logit transformation. Ordinate B/Be, on logit-ecale (33, antibody bound labelled cholic acid in cpm; 80, antibody bound labelled cholic acid in cpm at zero dose unlabelled glycocholic acid: N, non specific bound labelled cholie acid in cpmf.
were analysed by gas-liquid chromato~aphy Hi-Eff 8BP [ 17 f as stationary phase.
as described previously [ 161 using
Results Antiserum
The antigenic response of the six immunized animals was determined by Ouchterlony double-diffusion technique and by dilution of the serum that would bind 50% of 3000 cpm [ 3H]cholic acid. Five weeks after the beginning of the immunization, the serum obtained from each of the six animals mixed with an excess of bovine serum albumin showed in Ou~h~rlony double-dif~sion technique no pr~ipi~tion by diffusion against bovine serum albumin, whereas a single precipitation line was obtained by diffusion against cholic acidbovine serum albumin conjugate (Fig. 2). This indicates the presence of antibodies against the cholic acid in each of the immunized animals. Titers between l/200 and l/600 were found (example Fig. 3). Specificity
The specificity of the antiserum was investigated by adding other common bile acids to a solution containing labelled cholic acid and antiserum. The per cent cross reaction was calculated from the ratio of the mass of glycocholic acid required to displace 50% of labelled bound cholic acid to the mass of cross reacting bile acid required, m~tiplied by 100. Ta~ocholic acid was equiv~ent to glycocholic acid (Fig. 4). The percentage cross reaction of the antiserum R44 with common bile acids is given in Table I.
42
Fig. 2. Ouchterlony double-diffusion of bovine serum albumin and cholic acid-bovine serum albumin conjugate against antiserum to cholic acid. 0.3 ml of antiserum of rabbit R-44 to cholic acid treated with 100 /.~g of bovine serum albumin in the center well reacted with 100 pg of cholic acid-bovine serum albumin conjugate dissolved in 100 ~1 of waterlpyridine (30 : 1. v/v). BSA, 100 c(g of bovine serum albumin dissolved in 100 ~1 of waterlpyridine (30 : 1. v/v). The plate was developed for 3 days at 25°C.
Sensitivity and precision The sensitivity of the assay calculated from the standard curve, which was linear between 5 and 80 pmol of glycocholic acid with glyco[ ‘Hlcholic acid or [ 3H]cholic acid (Fig. 1) as labelled bile acid, would allow the determination of 0.05 I.tmol/l of conjugated cholic acid as the least detectable serum concentration by using 0.1 ml of undiluted serum in the assay procedure. Blank values obtained from water and from bile acid free serum were not different from 0. The within-assay precision at a low serum concentration of conjugated cholic acid (0.4 E.tmol/.l) was 13% and at a higher serum concentration (37 E.cmol/l) 14.4% (coefficient of variation). The between- assay precision determined by carrying a sample from a serum pool (0.7 pmol/l) in 6 different assays was 16.7% (coefficient of variation). The difference between serum values of conjugated cholic acid obtained with [ ‘Hlcholic acid (Fig. 1) or with glyco [ 3H]cholic acid as labelled bile acid used in the standard curve and the assay was within the within-assay precision. Accuracy The recovery of unlabelled glycocholic acid was estimated from 5 dose levels added to bile acid free serum and separated by Amberlite XAD-2. Aliquots containing 5, 10, 20, 40 and 80 pmol of glycocholic acid were assayed. The dis-
43
t 50 Dilution
200 of
800
antiserum
3200 R-44
(11~)
Fig. 3. Titration curve of I bound C3H] cholic acid plotted against dilution of antiserum from rabbit R44. (All assays included in this paper were performed with this antiserum in a final concentration of l/600).
GCDC
\\
5 DOS.
blla
acld
20
80
320
1280
b
(pmolas)
Fig. 4. Cross reaction of the cholic acid antiserum R-44 with other common bile acids. Ordinate: antibody bound labelled [3H]cholic acid as per cent of cpm bound in the absence of unlabelled ligand. TC, taurocholic acid; GC, glycocholic acid: C, cholic acid; GCDC, glycochenodeoxycholic acid: CDC, chenodeoxycholic acid; GDC, glycodeoxycholic acid; TDC. taurodeoxycholic acid; DC, deoxycholic acid; TLC, taurolithocholic acid; GLC, glycolithocholic acid; LC. lithocholic acid.
44 TABLE I CROSS REACTION
OF VARIOUS
BILE ACIDS WITH ANTISERUM
Bile acid
% Cross reaction
TauroehoIie Glycocholic Cholic Glycochenodeoxychoiie Chenodeoxyeholic Taurodeoxycholic Giycodeoxyeholic Deoxycholic Taurolithocholic Glycolithocholic Lithochoiic
100 100 56 13 5 3 3 <1
R-44
placement curve obtained was equivalent to the standard curve between 5 and 80 pmol. The ratio of glycocholic acid found to that added to the bile acid free serum was 103.8 * 3.9% (S.D.). The recovery of unlabelled taurocholic acid from serum was estimated in the same way as glycocholic acid. The ratio of taurocholic acid found to that added to the bile acid free serum was 101.2 + 6.1% (S.D.), Serum values Serum values for conjugated cholic acid in 8 normal fasting men, age 25-35 years, are shown in Table II and compared with data obtained from the gasliquid chromatography. The mean value determined by radioimmunoassay was 0.68 * 0.34 (S.D.), range 0.18 to 1.25 pmol/l. A good a~eement with values determined by gas-liquid chromatography was obtained (Table II). Values of serum conjugated cholic acid from 31 subjects with or without liver disease plotted against serum bilirubin as parameter for the degree of cholestasis are shown in Fig. 5. Serum values of 5 patients with different liver diseases and one patient with chronic pancreatitis before and at different times after endoscopic
TABLE II COMPARISON DETERMINED Sample No.
OF SERUM LEVELS OF CONYUGATED CHOLXC ACID IN NORMAL FASTING MEN BY RADIOIMMU~OASSAY AND BY GAS-LIQUID CHROMATOGRAPHY (GLC) Serum conjugated cholie acid @moI!B Radioimmunoassay
GLC *
1.25 0.96 0.82 0.46 0.61 0.46 0.18 0.76
1.02 1.26 1.10 0.38 0.79
* If present in serum, free choiic acid is inc‘luded. -,
indicates that bile acids were not determined.
45
+
.
I
70
I
I I
50
.
.
.
I
I
:
30
I I
“E = 10 Y u ._ : ; ._ c i3 Q F z s
5
.
I
.
I
I I
1 ‘.I
I I
4
I
I . 1 . ’ 0. I. 2 l__ !- ____ .*I 1 %, ‘*S .=J0. 1
____
2
3
Serum
4
_-----es--
5
6
bilirubin
(mg
7
8
X)
Fig. 5. Relationship of serum conjugated cholic acid to serum blllrubln levels for 31 subjects. Vertical broken line represents upper limit of normal for serum blllrubln; horizontal broken line represents upper limit of normal for serum bile acid.
TABLE
III
CLINICAL AND X-RAY DATA RAPHY (ERCP) IN PATIENTS
AT
ENDOSCOPIC
RETROGRADE
CHOLANGIOPANCREATOG-
Patient : &e. sex
Indication for ERCP
Opacifled ducts *
ERCP dlagnosls **
IS. : 63. F B.B. : 64. F
Cholestasls
p. c
Cholestasis
P. C
B.R. : 41. M K.M. : 21. F
Hepatomegaly
P, C
Raised erytbocyte sedimentation rate
P. C
Normal PI); normal EBT; normal IBT Normal PD; cholcdochollthlasls with free floating stone and dilated EBT and dilated IBT Normal PD; normal EBT; no IBT Normal PD; normal EBT: local dilation of IBT
F.S. : 53. M E.F.: 60. M
Abdominal pain after cholecystectomy Abdominal pain in alcohbollc
P P
Chronic pancrcatitls: no EBT: no IBT Normal PD; no EBT; no IBT
Clinical diagnosis
blliary clrrhosls Obstructive jaundice due to choledochollthlasis
Primary
Chronic cholangitis Unspecific liver damage (liver histology) Chronic pancreatitls Fatty liver
* P, pancreatography; C. cholangiography. ** PD. pancreatic duct; EBT. extrahepatic blllary tree; IBT. intrahepatic blliary tree.
46 1
ERCP
8.R. K.M. F.S. E.F. U’
-2
-1
0
+l
+2 time
+3
+4
+5
+18
(hours)
Fig. 6. Concentration of conjugated cholic acid in serum samples of 6 patients (for clinical data see Table III) taken before and at different times after endoscopic retrograde cholangiopancreatography (ERCP). Each value is the mean S.D. of two or three different dilutions of the extracted bile acids assayed in duplicate by radioimmunoassay.
retrograde Fig. 6.
cholangiopancreatography
(clinical data see Table III) are shown in
Discussion Recently Simmonds et al. [6] and Murphy et al. [7] reported a radioimmunoassay for serum conjugated cholic acid using an antiserum obtained from rabbits immunized with glycocholic acid-bovine serum albumin conjugate. These antibodies had little affinity for unconjugated cholic acid, as 25 mol [6] or 10 mol [7] of free cholic acid were required to displace 1 molecule of taurocholic or glycocholic acids. The present antiserum obtained after immunization with cholic acid-bovine serum albumin conjugate showed a predominant affinity to conjugated cholic acid (Fig. 4) but only two molecules of free cholic acid were required to displace 1 molecule of taurocholic or glycocholic acids (Table I). Therefore values determined by the present radioimmunoassay seem not only include glycine and taurine conjugated cholic acid but possibly also free cholic acid. However the good agreement of serum values in healthy persons determined by the present radioimmunoassay (Table II) (range 0.1% 1.25 E.tmol/l) with the serum values of conjugated cholic acid determined by Simmonds et al. [6] (range 0.2-1.0 I.tmol/l) and by Murphy et al. [7] (range 0.55-1.8 pmol/l) shows that in healthy persons, free cholic acid seems to be only a small part of the serum total cholic acid. This is in agreement with gas-
47
liquid chromatography data published by Sandberg et al. [l] and by Makino et al. [18] who described free cholic acid in serum of healthy persons and calculated that free cholic acid serum concentration is about half that of conjugated cholic acid [IS]. Prior to the radioimmunoassay, extraction of serum bile acids was performed using Amberlite XAD-2 as described by Makino and Sjijvall [5] as bile acids are bound to albumin in plasma and binding is decreased at high pH [19]. That values of conjugated cholic acid determined by radioimmuno~say obtained in unextracted sera increased with increasing dilution was shown by Murphy et al. [ 71. Therefore an extraction of serum bile acids seems to be necessary. The agreement between values obtained with the present radioimmunoassay and gas-liquid chromatography (Table II) suggests the reliability of the radioimmunoassay for determination of serum conjugated cholic acid possibly including free cholic acid. However, the interference of conjugated dihydroxy bile acids must be taken into consideration, if present in greater proportion than conjugated cholic acid (Table I). The sensitivity and the practicability of this assay recommend the usefulness of this method for looking at bile acids in health and disease. Recently Hofmann [ 203 showed that clearance of intravenous injected glycocholic acid, determined by ~dioimmuno~say, seems to be a more sensitive index of liver function than only fasting serum conjugated cholic acid (Fig. 5). However in interpretation of serum bile acids, meals [21] and diagnostic methods i.e. endoscopic retrograde cholangiopancreatography (Fig. 6), which may influence the serum conjugated cholic acid levels, should be taken into account. Acknowledgement This work was supported No. Ma 567/2).
by the Deutsche
Forschungsgemeinschaft
(grant
References 1 Sandberg. D.H., SiiivaII, J., Sjavall, K. and Turner, D.A. (1965) J. Lipid Res. 6, 182-192 2 PanveIiwaIla, D.. Lewis, B., Wootton, I.D.P. and. TabaqchaIi. S. (1970) J. Clin. Pathol. 23, 309314 3 Roovers, J., Evrard, E. and Vanderhaeghe, H. (1968) Clin. Chim. Acta 19, 449-457 4 AIi, S.S. and Javitt, N.B. (1970) Can. J. Biochem. 48.1054-1057 5 Makino, 1. and SjijvaII, J. (1972) AnaI. Lett. 5.341-349 6 Simmonds, W.J., Korman, M.G., Go, V.L.W. and Hofmann. A. F. (1973) Gastroenterology 65.705711 7 Murphy, G.M., Edkins, S.M., ~ilIiams, J.W. and Catty, D. (1974) CIin. Chim. Acta 64.81-89 6 Klopstock, A., Pinto, M. and Rimon. A. (1964) J. Immunol. 92, 516-619 9 Erlanger. B.F.. Borek, F.. Beiser. S.M. and Lieberman, S. (1957) J. Biol. Chem. 228. 713-727 10 Cronhohn, T., Eriksson, H.. Matern. S. and SjiivalI, J. (1976) Eur. J. Biochem. 53.406-410 11 Lowry, O.K., Rosabrough, NJ., Farr, A.L. and Randall, RI. (1951) J. BioI. Chem. 193.266-275 12 Matem, S., FrBhIIng, W. and Bock, K.W. (1972) Naunyn-Sch~edeberg’s Arch. Pharmacoi. 273, 242-247 13 14 15 16
Bock. K.W. and Matem. S. (1973) Eur. J. Biochem. 38.20-24 Cronhohn, T., Makino. I. and SjisvaII. J. (1972) Eur. J. Biochem. 26.251-268 CronhoIm, T.. Makino, I. and Sj&aII. J. (1972) Eur. J. Biochem. 24, 507-519 Matern. S., SjiivaII, J.. Pomare, E.W., Heaton. K.W. and Low-Beer, T.S. (1975) Med. Biology 63,107113 17 Makita, M. and WeBs, W.W. (1963) Anal. Biochem. 6.523-530
48 18 Makino, I., Nakagawa, S. and Mashimo. K. (1969) Gastroenterology 56. 1033-1039 19 Rudman, D. and Kendall, F.E. (1957) J. Clin. Invest, 36. 530-537 20 Hofmann, A.F. (1975) in Advances in Bile Acid Research (Matern, S.. Hackenschmidt. and Gerok, W., eds.). pp. 245-247, F.K. Schattauer Verlag. Stuttgart-New York 21 LaRusso, N.F.. Korman. M.G.. Hoffman, N.E. and Hofmann, A.F. (1974) N. En& 689-692
J.. Back, P. J. Med.
291,
Clinica Chimica Acta, 72 (1976) 39-48 @ Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
CCA 7894
RADIO~UNOASSAY
SIEGFRIED
OF SERUM CONJUGATED CHOLIC ACID
MATERN *, RUTH KRIEGER
and WOLFGANG GEROK
Medizinische Kfinik der Universitiit Freiburg i.Br. (G. F. R.) (Received February 18th, 1976)
Summary A radioimmunoassay for serum conjugated cholic acid is described using antiserum obtained five weeks after immunization of rabbits with cholic acidbovine serum albumin conjugate. Prior to the radioimmunoassay, extraction of bile acids was performed with Amberlite XAD-2. The displacement curve of glyco[ 3H]cholic acid was linear on a log&log plot from 5 to 80 pmol of unlabelled glycocholic acid. Values for 8 normal fasting subjects ranged from 0.18 to 1.25 E.tmol/l. In 31 fasting subjects with or without liver disease serum values of conjugated cholic acid are related to serum bilirubin. Endoscopie retrograde cholangiopancreatography may influence serum levels of conjugated cholic acid.
Introduction Methods for determination of individual bile acids in serum are usually based on gas-liquid or thin-layer chromatography [l-5]. However, gas-liquid chromatography is time consuming and thin-layer chromato~aphy insensitive. Only recently a sensitive radioimmunoassay for determination of serum conjugated bile acids was first developed by Simmonds et al. [6] and by Murphy et al. [ 71, about ten years after the first report of production of antiserum against cholanic arid-~bumin conjugates 181. This paper describes a radioimmuno~~y, of serum conjugated cholic acid with an antiserum obtained after immunization of rabbits with cholic acid-bovine serum albumin conjugate. Materials and methods Unlabelled bile acids were obtained from Steraloids Ltd. [2,43H]Cholic acid with a specific activity of 0.25 mCi/0.03’7 mg, glycof 3H]cholie acid with a spe* Address correspondence to: Dr. Siegfried Matern, Ned&in&he 56. D-78 Freiburg i.Br., G.F.R.
UniversitWkJinik,
Hugstetter Str.
40
cific activity of 0.25 mCi/0.039 mg and tauro[3H]cholic acid with a specific activity of 0.25 mCi/0.038 mg were obtained from New England Nuclear, Boston, Mass. The scintillation liquid was Unisolve 1 obtained from Koch-Light Ltd., England. Liquid scintillation counting was done in a Packard Tricarb scintillations spectrometer, model 3380. Complete Freund’s adjuvant was supplied by the Difco Laboratories. Polyethylene glycol and Amberlite XAD-2 were purchased from Serva (Heidelberg), Sephadex LH-20 from Pharmacia (Uppsala/Sweden), bovine serum albumin from Beringwerke (Marburg) and HiEff 8BP from Applied Science Laboratories, State College, Pennsylvania. All reagents (Merck AG, Darmstadt) were of analytical grade and all solvents (Merck AG, Darmstadt) were redistilled. Preparation of antigen Cholic acid was rendered antigenic by coupling to bovine serum albumin with a mixed anhydride reaction as described by Erlanger et al. [9] and by Murphy et al. [ 71. The cholic acid-bovine serum albumin conjugate contained 12 mol of bile acid per mol albumin determined by gas-liquid chromatography [lo]. Protein was determined by the method of Lowry et al. [ 111. Immunization Antiserum was obtained after 4 weekly injections of 1 mg of cholic acidbovine serum albumin conjugate emulsified with Freund’s complete adjuvant into the dorsal skin in each of 6 female rabbits as described previously [ 12,131. Radioimmunoassay Prior to the radioimmunoassay extraction of bile acids was performed by passing 1 ml of serum through a column of Amberlite XAD-2 as described by Makino and Sjijvall [ 51. The evaporated eluate from Amberlite XAD-2 was reconstituted to a volume of 1 ml with ethanol and diluted with 0.01 M potassium phosphate buffer, pH 7.4 to 5 ml. Aliquots of OJ ml were assayed. The radioimmunoassay was performed as described by Simmonds et al. [6] with the following minor modifications: In each tube of the assay were placed either 8000 cpm glyco [ 3H]cholic acid or 8000 cpm [ 3H]cholic acid and in each tube of the standard curve was ethanol in a dilution of 1 : 50. Standard curves obtained with or without this ethanol dilution were identical. The sample values were extrapolated from a straight line standard curve, which was obtained by plotting logit of (B--N)/(B,--N) X 100 against the logarithm of the dose (B, antibody bound labelled bile acid in cpm; BO, antibody bound labelled bile acid in cpm at zero dose unlabelled glycocholic acid; N, non specific bound labelled bile acid in cpm). The standard curve was linear between 5 and 80 pmol of unlabelled glycocholic acid with glyco [ 3H]cholic acid as well as with [ 3H]cholic acid (example Fig. 1) as labelled bile acid. Gas-liquid chromatography of serum nonsulphated bile acids After extraction of serum bile acids with Amberlite XAD-2 [5] the non-sulphated bile acids separated on Sephadex LH-20 as described by Cronholm et al. [ 141 were hydrolysed and the methyl ester trimethylsilyl ether derivatives [ 151
41
DOS.
glycochollc
rcld
(pmOl@S)
Fig. 1. Standard curve in logit transformation. Ordinate B/Be, on logit-ecale (33, antibody bound labelled cholic acid in cpm; 80, antibody bound labelled cholic acid in cpm at zero dose unlabelled glycocholic acid: N, non specific bound labelled cholie acid in cpmf.
were analysed by gas-liquid chromato~aphy Hi-Eff 8BP [ 17 f as stationary phase.
as described previously [ 161 using
Results Antiserum
The antigenic response of the six immunized animals was determined by Ouchterlony double-diffusion technique and by dilution of the serum that would bind 50% of 3000 cpm [ 3H]cholic acid. Five weeks after the beginning of the immunization, the serum obtained from each of the six animals mixed with an excess of bovine serum albumin showed in Ou~h~rlony double-dif~sion technique no pr~ipi~tion by diffusion against bovine serum albumin, whereas a single precipitation line was obtained by diffusion against cholic acidbovine serum albumin conjugate (Fig. 2). This indicates the presence of antibodies against the cholic acid in each of the immunized animals. Titers between l/200 and l/600 were found (example Fig. 3). Specificity
The specificity of the antiserum was investigated by adding other common bile acids to a solution containing labelled cholic acid and antiserum. The per cent cross reaction was calculated from the ratio of the mass of glycocholic acid required to displace 50% of labelled bound cholic acid to the mass of cross reacting bile acid required, m~tiplied by 100. Ta~ocholic acid was equiv~ent to glycocholic acid (Fig. 4). The percentage cross reaction of the antiserum R44 with common bile acids is given in Table I.
42
Fig. 2. Ouchterlony double-diffusion of bovine serum albumin and cholic acid-bovine serum albumin conjugate against antiserum to cholic acid. 0.3 ml of antiserum of rabbit R-44 to cholic acid treated with 100 /.~g of bovine serum albumin in the center well reacted with 100 pg of cholic acid-bovine serum albumin conjugate dissolved in 100 ~1 of waterlpyridine (30 : 1. v/v). BSA, 100 c(g of bovine serum albumin dissolved in 100 ~1 of waterlpyridine (30 : 1. v/v). The plate was developed for 3 days at 25°C.
Sensitivity and precision The sensitivity of the assay calculated from the standard curve, which was linear between 5 and 80 pmol of glycocholic acid with glyco[ ‘Hlcholic acid or [ 3H]cholic acid (Fig. 1) as labelled bile acid, would allow the determination of 0.05 I.tmol/l of conjugated cholic acid as the least detectable serum concentration by using 0.1 ml of undiluted serum in the assay procedure. Blank values obtained from water and from bile acid free serum were not different from 0. The within-assay precision at a low serum concentration of conjugated cholic acid (0.4 E.tmol/.l) was 13% and at a higher serum concentration (37 E.cmol/l) 14.4% (coefficient of variation). The between- assay precision determined by carrying a sample from a serum pool (0.7 pmol/l) in 6 different assays was 16.7% (coefficient of variation). The difference between serum values of conjugated cholic acid obtained with [ ‘Hlcholic acid (Fig. 1) or with glyco [ 3H]cholic acid as labelled bile acid used in the standard curve and the assay was within the within-assay precision. Accuracy The recovery of unlabelled glycocholic acid was estimated from 5 dose levels added to bile acid free serum and separated by Amberlite XAD-2. Aliquots containing 5, 10, 20, 40 and 80 pmol of glycocholic acid were assayed. The dis-
43
t 50 Dilution
200 of
800
antiserum
3200 R-44
(11~)
Fig. 3. Titration curve of I bound C3H] cholic acid plotted against dilution of antiserum from rabbit R44. (All assays included in this paper were performed with this antiserum in a final concentration of l/600).
GCDC
\\
5 DOS.
blla
acld
20
80
320
1280
b
(pmolas)
Fig. 4. Cross reaction of the cholic acid antiserum R-44 with other common bile acids. Ordinate: antibody bound labelled [3H]cholic acid as per cent of cpm bound in the absence of unlabelled ligand. TC, taurocholic acid; GC, glycocholic acid: C, cholic acid; GCDC, glycochenodeoxycholic acid: CDC, chenodeoxycholic acid; GDC, glycodeoxycholic acid; TDC. taurodeoxycholic acid; DC, deoxycholic acid; TLC, taurolithocholic acid; GLC, glycolithocholic acid; LC. lithocholic acid.
44 TABLE I CROSS REACTION
OF VARIOUS
BILE ACIDS WITH ANTISERUM
Bile acid
% Cross reaction
TauroehoIie Glycocholic Cholic Glycochenodeoxychoiie Chenodeoxyeholic Taurodeoxycholic Giycodeoxyeholic Deoxycholic Taurolithocholic Glycolithocholic Lithochoiic
100 100 56 13 5 3 3 <1
R-44
placement curve obtained was equivalent to the standard curve between 5 and 80 pmol. The ratio of glycocholic acid found to that added to the bile acid free serum was 103.8 * 3.9% (S.D.). The recovery of unlabelled taurocholic acid from serum was estimated in the same way as glycocholic acid. The ratio of taurocholic acid found to that added to the bile acid free serum was 101.2 + 6.1% (S.D.), Serum values Serum values for conjugated cholic acid in 8 normal fasting men, age 25-35 years, are shown in Table II and compared with data obtained from the gasliquid chromatography. The mean value determined by radioimmunoassay was 0.68 * 0.34 (S.D.), range 0.18 to 1.25 pmol/l. A good a~eement with values determined by gas-liquid chromatography was obtained (Table II). Values of serum conjugated cholic acid from 31 subjects with or without liver disease plotted against serum bilirubin as parameter for the degree of cholestasis are shown in Fig. 5. Serum values of 5 patients with different liver diseases and one patient with chronic pancreatitis before and at different times after endoscopic
TABLE II COMPARISON DETERMINED Sample No.
OF SERUM LEVELS OF CONYUGATED CHOLXC ACID IN NORMAL FASTING MEN BY RADIOIMMU~OASSAY AND BY GAS-LIQUID CHROMATOGRAPHY (GLC) Serum conjugated cholie acid @moI!B Radioimmunoassay
GLC *
1.25 0.96 0.82 0.46 0.61 0.46 0.18 0.76
1.02 1.26 1.10 0.38 0.79
* If present in serum, free choiic acid is inc‘luded. -,
indicates that bile acids were not determined.
45
+
.
I
70
I
I I
50
.
.
.
I
I
:
30
I I
“E = 10 Y u ._ : ; ._ c i3 Q F z s
5
.
I
.
I
I I
1 ‘.I
I I
4
I
I . 1 . ’ 0. I. 2 l__ !- ____ .*I 1 %, ‘*S .=J0. 1
____
2
3
Serum
4
_-----es--
5
6
bilirubin
(mg
7
8
X)
Fig. 5. Relationship of serum conjugated cholic acid to serum blllrubln levels for 31 subjects. Vertical broken line represents upper limit of normal for serum blllrubln; horizontal broken line represents upper limit of normal for serum bile acid.
TABLE
III
CLINICAL AND X-RAY DATA RAPHY (ERCP) IN PATIENTS
AT
ENDOSCOPIC
RETROGRADE
CHOLANGIOPANCREATOG-
Patient : &e. sex
Indication for ERCP
Opacifled ducts *
ERCP dlagnosls **
IS. : 63. F B.B. : 64. F
Cholestasls
p. c
Cholestasis
P. C
B.R. : 41. M K.M. : 21. F
Hepatomegaly
P, C
Raised erytbocyte sedimentation rate
P. C
Normal PI); normal EBT; normal IBT Normal PD; cholcdochollthlasls with free floating stone and dilated EBT and dilated IBT Normal PD; normal EBT; no IBT Normal PD; normal EBT: local dilation of IBT
F.S. : 53. M E.F.: 60. M
Abdominal pain after cholecystectomy Abdominal pain in alcohbollc
P P
Chronic pancrcatitls: no EBT: no IBT Normal PD; no EBT; no IBT
Clinical diagnosis
blliary clrrhosls Obstructive jaundice due to choledochollthlasis
Primary
Chronic cholangitis Unspecific liver damage (liver histology) Chronic pancreatitls Fatty liver
* P, pancreatography; C. cholangiography. ** PD. pancreatic duct; EBT. extrahepatic blllary tree; IBT. intrahepatic blliary tree.
46 1
ERCP
8.R. K.M. F.S. E.F. U’
-2
-1
0
+l
+2 time
+3
+4
+5
+18
(hours)
Fig. 6. Concentration of conjugated cholic acid in serum samples of 6 patients (for clinical data see Table III) taken before and at different times after endoscopic retrograde cholangiopancreatography (ERCP). Each value is the mean S.D. of two or three different dilutions of the extracted bile acids assayed in duplicate by radioimmunoassay.
retrograde Fig. 6.
cholangiopancreatography
(clinical data see Table III) are shown in
Discussion Recently Simmonds et al. [6] and Murphy et al. [7] reported a radioimmunoassay for serum conjugated cholic acid using an antiserum obtained from rabbits immunized with glycocholic acid-bovine serum albumin conjugate. These antibodies had little affinity for unconjugated cholic acid, as 25 mol [6] or 10 mol [7] of free cholic acid were required to displace 1 molecule of taurocholic or glycocholic acids. The present antiserum obtained after immunization with cholic acid-bovine serum albumin conjugate showed a predominant affinity to conjugated cholic acid (Fig. 4) but only two molecules of free cholic acid were required to displace 1 molecule of taurocholic or glycocholic acids (Table I). Therefore values determined by the present radioimmunoassay seem not only include glycine and taurine conjugated cholic acid but possibly also free cholic acid. However the good agreement of serum values in healthy persons determined by the present radioimmunoassay (Table II) (range 0.1% 1.25 E.tmol/l) with the serum values of conjugated cholic acid determined by Simmonds et al. [6] (range 0.2-1.0 I.tmol/l) and by Murphy et al. [7] (range 0.55-1.8 pmol/l) shows that in healthy persons, free cholic acid seems to be only a small part of the serum total cholic acid. This is in agreement with gas-
47
liquid chromatography data published by Sandberg et al. [l] and by Makino et al. [18] who described free cholic acid in serum of healthy persons and calculated that free cholic acid serum concentration is about half that of conjugated cholic acid [IS]. Prior to the radioimmunoassay, extraction of serum bile acids was performed using Amberlite XAD-2 as described by Makino and Sjijvall [5] as bile acids are bound to albumin in plasma and binding is decreased at high pH [19]. That values of conjugated cholic acid determined by radioimmuno~say obtained in unextracted sera increased with increasing dilution was shown by Murphy et al. [ 71. Therefore an extraction of serum bile acids seems to be necessary. The agreement between values obtained with the present radioimmunoassay and gas-liquid chromatography (Table II) suggests the reliability of the radioimmunoassay for determination of serum conjugated cholic acid possibly including free cholic acid. However, the interference of conjugated dihydroxy bile acids must be taken into consideration, if present in greater proportion than conjugated cholic acid (Table I). The sensitivity and the practicability of this assay recommend the usefulness of this method for looking at bile acids in health and disease. Recently Hofmann [ 203 showed that clearance of intravenous injected glycocholic acid, determined by ~dioimmuno~say, seems to be a more sensitive index of liver function than only fasting serum conjugated cholic acid (Fig. 5). However in interpretation of serum bile acids, meals [21] and diagnostic methods i.e. endoscopic retrograde cholangiopancreatography (Fig. 6), which may influence the serum conjugated cholic acid levels, should be taken into account. Acknowledgement This work was supported No. Ma 567/2).
by the Deutsche
Forschungsgemeinschaft
(grant
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Bock. K.W. and Matem. S. (1973) Eur. J. Biochem. 38.20-24 Cronhohn, T., Makino. I. and SjisvaII. J. (1972) Eur. J. Biochem. 26.251-268 CronhoIm, T.. Makino, I. and Sj&aII. J. (1972) Eur. J. Biochem. 24, 507-519 Matern. S., SjiivaII, J.. Pomare, E.W., Heaton. K.W. and Low-Beer, T.S. (1975) Med. Biology 63,107113 17 Makita, M. and WeBs, W.W. (1963) Anal. Biochem. 6.523-530
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