247
Clinica Chimica Acta, 99 (1979) 247-251 @ Elsevier/North-Holland Biomedical Press
CCA 1181
AN EASY PROCEDURE FOR DETERMINATION OF MOLAR ACTIVITY (OR SPECIFIC ACTIVITY) OF BILE ACIDS IN BILE
ARNE BRUUSGAARD, Department
HANNA SQRENSEN and JESPER SYLVEST
of Clinical Chemistry, Frederiksberg
Hospital, Copenhagen
(Denmark)
(Received May 31st, 1979)
Summary An easy procedure is described for determination of the molar activity of the major bile acids, labelled with 14C, in bile. The procedure involves initial enzymatic hydrolysis, by which the amino acid moieties are removed from the glycine and taurine conjugated bile acids by means of choloylglycine hydrolase, followed by thin-layer chromatographic separation of the unconjugated bile acids, cholic acid, chenodeoxycholic acid, and deoxycholic acid. Then the bile acids are eluted from the individual silica gel spots concerned. Finally, determination of the radioactivity by liquid scintillation counting and of the amount of substance by an enzymatic method using 3a-hydroxysteroid dehydrogenase is performed in the eluates. The method requires only a small volume of sample and allows of separate determination of the molar activity of cholic acid and chenodeoxycholic acid, labelled with the same isotope, in the same sample.
Introduction Evaluation of the size and kinetics of the bile acid pool, especially for the major bile acids, cholic acid and chenodeoxycholic acid, has become of increasing interest in the gastrointestinal and hepatobiliary field. The most commonly used methods are based on determination of the dilution of an administered tracer-labelled (3H, or preferably 14C) bile acid after mixing with the endogenous pool [l-3], i.e. based on the measurement of the molar activity in bile of the bile acid concerned (molar activity is more informative than specific activity, especially when comparing interrelated substances of different relative molecular mass). The molar activity is defined as radioactivity divided by amount of substance (unit: Bq/mol). Correspondence should be addressed to: Dr. Ame Bruusgaard. Department of Clinical Chemistry. Frederiksberg Hospital, Nordre Fasanvej 69. DK-2000 Copenhagen F, Denmark.
248
Current methods for this determination often involve cumbersome steps such as gas-liquid chromatography [ 3-51, alkaline hydrolysis of the conjugated bile acids [ 3+], or repeated thin-layer chromatography [ 21. A drawback of some published procedures is a lack of information on the volume of sample required for the analysis. This is the case in the papers of Duane et al. [3], Vlahcevic et al. [4], and Einarsson et al. [ 51. They only state the volume of duodenal sample retained during aspiration, and this volume and thereby the amount of bile acid removed from the organism - seems to be rather high, implying the risk of induction of significant changes in the physiological steady state of the bile acid metabolism. Here a simplified procedure is described for the determination of molar activity of bile acids in bile, based on conventional methods, requiring only a small volume of sample and facilities available in many hospital laboratories. The method allows separate determination of the molar activities of cholic acid and chenodeoxycholic acid in the same sample, even when these bile acids are labelled with the same isotope. Material and methods Samples Duodenal aspirates were obtained after stimulation of the gallbladder emptying with 40-80 Ivy dog units of cholecystokinin (CCK-PZ, GIH Research Unit, Chemistry Department, Karolinska Institutet, Stockholm, Sweden) injected i.v. An aliquot of 0.5-1.5 ml of the volume aspirated at each time was retained for analysis. The aspirates were obtained at various intervals after i.v. administration of approximately 3.7 X 10’ Bq = 10 &i carboxy-i4C-cholate and 3.7 X 10’ Bq = 10 $!i carboxy-14C-chenodeoxycholate (The Radiochemical Centre, Amersham, U.K.). Choloylglycine hydrolase (EC 3.5.1.24) solution A solution of 15 mg of Clostridium welchii acetone powder, Type III (Sigma No. C-8635) per 1 ml of disodium hydrogen phosphate buffer, 0.2 mol/l, pH 5.8, was prepared. Thorough mixing for 5 min was followed by centrifugation at 2000 X g for 20 min. The supernatant contains the enzyme, capable of hydrolyzing glycine as well as taurine conjugates of bile acids. do-Hydroxysteroid dehydrogenase (3a-HSD) (EC 1 .l. 1.50) Sterognost-3cy@ from Nyegaard and Co. A/S, Oslo, Norway. Liquid scintillation counting solution Naphthalene 60 g, PPO (2,5-diphenyl-1,3-oxazole) 4 g, POPOP (1,4-bis( 2-( 5phenyl-oxazolyl))-benzene) 0.2 g, methanol 100 ml, ethylene glycol 20 ml, p-dioxane ad 1000 ml. Procedure Initially, the total bile acid concentration mined enzymatically (3ol-HSD) as previously
in the duodenal described [ 71.
sample was deter-
249
An appropriate volume (usually 100-300 ~1) of the sample, containing about 4 pmol of total bile acid, was subjected to enzymatic hydrolysis by mixing with 4 ml of the choloylglycine hydrolase solution and incubation of the mixture for 30 min in a water-bath at 37°C under constant shaking. After cooling to room temperature the pH of the mixture was adjusted to approximately 1 (pH-indicator paper) with 6 mol/l HCl. The deconjugated bile acids were then extracted three times with 2 ml of diethyl ether. The pooled ether extracts were evaporated to dryness at room temperature under a stream of nitrogen. The residue was redissolved in 100 ~1 of methanol and applied to a thin-layer plate with silica gel [ $1 in a 30 mm broad band, after which the chromatogram was developed according to previous description [ 81. In this way a satisfactory separation of the deconjugated bile acids, cholic acid, chenodeoxycholic acid, and deoxycholic acid, was obtained, although the latter two were situated rather near to each other. With the aid of reference solutions run in parallel on the same plate, the spots were detected by exposing the plate to iodine vapor and delineated. Thereafter, the iodine was allowed to sublimate at room temperature. The delineated bands with the individual bile acids were scraped off, in the case of chenodeoxycholic acid taking precautions to avoid the verges of the band. The removed silica gel was transferred to a glass-sintered column, and the bile acid was eluted with 15 ml of methanol. The methanol solution was evaporated to dryness on a water-bath at 7080°C under a stream of nitrogen, and the residue was redissolved in 2 ml of methanol. Of this volume, 1 ml (+ 10 ml of the counting solution) was taken for measurement of radioactivity in a Beckman liquid scintillation counter (LS 250), using external standardization to correct for quenching, and the remainder was used for determination of the concentration of bile acids by the 3a-HSD method (in duplicate) [ 71. The molar activity was calculated from the values obtained for activity concentration (Bq/l) and the substance concentration of bile acid (mol/l), the unit thus being Bq/mol. (The factor of conversion from the hitherto most commonly used unit “disintegrations per min/pmol” to Bq/mol is 1.67 X 104). Comparative method For comparison with the procedure described above, the molar activity of cholic acid was determined from the glycine and taurine conjugates of cholic acid. The sample was handled as previously stated [ 81, resulting in a chromatogram with separation of glycocholic acid and taurocholic acid from the dihydroxy bile acid conjugates. The removed silica gel spots with glycocholic acid and taurocholic acid, respectively, were pooled and thereafter followed the last part of the procedure described above.
250
Results and discussion The a~uly~~ca~ uariutio~ of the method was evaluated from the standard deviation for two totally independent results of duplicate analyses on different days of the same duodenal sample on 21 occasions (samples from five patients), the standard deviation being calculated from: d(EdF/2n). For cholic acid it was 6.6 X lo6 Bq/mol (interval of individual mean values 8.7 X 106-2.8 X 108, mean 1.1 X lo”), and for chenodeoxycholic acid 6.8 X lo6 Bq/mol (interval of individu~ mean values 2.6 X 107-1.9 X 108, mean 1.1 X 10X). Cholic acid was well separated from the other major bile acids, but as the spots of chenodeoxycholic acid and deoxycholic acid were situated rather near to each other, contamination of the removed chenodeoxycholic acid silica gel spot from the deoxycholic acid spot was a possibility, in spite of careful seraping of the central part of the spot. Rowever, the almost identical standard deviations {at the same level of molar activity) for cholic acid and chenodeoxycholic acid make it unlikely that significant contamination had occurred, as the possible contamination is unlikely to be of equal magnitude for the two scrapings of chenodeoxycholic acid for each result. Thus, it appears that the molar activity of both the major primary bile acids can be determined separately, even when labelled with the same isotope. The detection limit was taken as three times the standard deviation stated above, i.e. 19.8 X lo6 Bq/mol for cholic acid, and 20.4 X lo6 Bq/mol for chenodeoxycholic acid. The volume o~sarn~~e required for the analysis with the procedure described is small, which may be of decisive importance. If the Lindstedt technique [l] is used, involving serial determinations to obtain the molar activity elimination curve for the bile acids, the volume of duodenal sample (and thereby the amount of bile acids) retained for analysis should be as small as possible. In some of the current methods, the volume of sample retained is rather high, up to 10 ml of concentrated bile [5]. As the concentration of bile acids in duodenal aspirates after stimulation of the gallbladder emptying with cholecystokinin is usually 15-50 pmol/ml, a considerable amount of the bile acid pool (and isotope) is removed, significantly influencing the physiological steady state of the bile acid metabolism. For the ~~~~o~~sis of the conjugated bile acids an enzymatic method was used, which is in accordance with the recommendation to replace alkaline hydrolysis with an enzymatic procedure [9,10]. Furthermore, the enzymatic method is simpler. Our experiments showed that with 1 ml of the enzyme solution per 1 pmol of bile acid complete hydrolysis of glycine as well as of the taurine conjugates is achieved. A further advantage of the present method is that only the last steps in the procedure, the determination of radioactivity and of the bile acid concentration, need to be quantitative, and that the result of the analysis may be available in about 5 h .
251
Comparison of two methods Comparison of the values for the molar activity of cholic acid in duodenal samples with the two methods described was performed in 45 instances (4-5 samples from each of 10 patients). Theoretically, the values found with the two methods should be identical, i.e. that the ratio of the two results in each pair was 1.00, or, in practice, that the ratios would be evenly distributed about 1.00. However, the Wilcoxon test for pair differences showed significantly higher results with the hydrolytic procedure than with the other method @ < 0.01). The ratios of the values obtained with the deconjugated cholic acid to the values derived from the glycine and taurine conjugated cholic acid were in the interval of 0.93 to 1.27, median value 1.07, and only 6 out of the 45 ratios were below 1.00. It is difficult to ascertain which of the two results is the true one. A reasonable explanation for the difference is that some 3cu-hydroxy bile acid other than glycocholic acid and taurocholic acid and with a lower molar activity (possibly zero) than that of these was scraped off from the plate together with glycocholic acid and taurocholic acid. This would increase the bile acid concentration in that eluate to a higher degree than the radioactivity concentration, if the contaminating bile acid after hydrolysis does not appear in the spot of the unconjugated cholic acid. References 1 2 3 4 5 6 7 8 9 10
Lindstedt, S. (1957) Acta Phys. Stand. 40. l-9 Pomare, E.W. and Low-Beer, T.S. (1974) CIin. Chim. Acta 57. 239-248 Duane, W.C., Adler. R.D.. Bennion. L.J. and Ginsberg, R.L. (1975) J. Lipid Res. 16. 155-158 Vlahcevic, Z.R.. Be& C.C. Jr., Buhac, I., Farrar. J.T. and Swell. L. (1970) Gastroenterology 59,165173 Einarsson, K., HeIIstrSm. K. and KaIIner, M. (1974) J. CIin. Invest. 54,1301-1311 Hepner, G.W. (1975) Gastroenterology 68,1574-1581 Bruusgaard, A., Sdrensen. H.. Gilhuus-Moe, C.-C. and Sk&egg. B.A. (1977) CIin. Chim. Acta 77. 387395 Bruusgaard. A. (1970) CIin. Chim. Acta 28.495-504 Roovers, J., Evrard, E. and Vanderhaeghe. H. (1968) CIin. Chim. Acta 19. 449457 Eneroth, P. and Sj6vaB. J. (1971) in The Bile Acids, Volume 1: Chemistry (Nair, P.P. and Kritchevsky, D., eds.). p. 133, Plenum Press, New York -London
Clinica Chimica Acta, 99 (1979) 247-251 @ Elsevier/North-Holland Biomedical Press
CCA 1181
AN EASY PROCEDURE FOR DETERMINATION OF MOLAR ACTIVITY (OR SPECIFIC ACTIVITY) OF BILE ACIDS IN BILE
ARNE BRUUSGAARD, Department
HANNA SQRENSEN and JESPER SYLVEST
of Clinical Chemistry, Frederiksberg
Hospital, Copenhagen
(Denmark)
(Received May 31st, 1979)
Summary An easy procedure is described for determination of the molar activity of the major bile acids, labelled with 14C, in bile. The procedure involves initial enzymatic hydrolysis, by which the amino acid moieties are removed from the glycine and taurine conjugated bile acids by means of choloylglycine hydrolase, followed by thin-layer chromatographic separation of the unconjugated bile acids, cholic acid, chenodeoxycholic acid, and deoxycholic acid. Then the bile acids are eluted from the individual silica gel spots concerned. Finally, determination of the radioactivity by liquid scintillation counting and of the amount of substance by an enzymatic method using 3a-hydroxysteroid dehydrogenase is performed in the eluates. The method requires only a small volume of sample and allows of separate determination of the molar activity of cholic acid and chenodeoxycholic acid, labelled with the same isotope, in the same sample.
Introduction Evaluation of the size and kinetics of the bile acid pool, especially for the major bile acids, cholic acid and chenodeoxycholic acid, has become of increasing interest in the gastrointestinal and hepatobiliary field. The most commonly used methods are based on determination of the dilution of an administered tracer-labelled (3H, or preferably 14C) bile acid after mixing with the endogenous pool [l-3], i.e. based on the measurement of the molar activity in bile of the bile acid concerned (molar activity is more informative than specific activity, especially when comparing interrelated substances of different relative molecular mass). The molar activity is defined as radioactivity divided by amount of substance (unit: Bq/mol). Correspondence should be addressed to: Dr. Ame Bruusgaard. Department of Clinical Chemistry. Frederiksberg Hospital, Nordre Fasanvej 69. DK-2000 Copenhagen F, Denmark.
248
Current methods for this determination often involve cumbersome steps such as gas-liquid chromatography [ 3-51, alkaline hydrolysis of the conjugated bile acids [ 3+], or repeated thin-layer chromatography [ 21. A drawback of some published procedures is a lack of information on the volume of sample required for the analysis. This is the case in the papers of Duane et al. [3], Vlahcevic et al. [4], and Einarsson et al. [ 51. They only state the volume of duodenal sample retained during aspiration, and this volume and thereby the amount of bile acid removed from the organism - seems to be rather high, implying the risk of induction of significant changes in the physiological steady state of the bile acid metabolism. Here a simplified procedure is described for the determination of molar activity of bile acids in bile, based on conventional methods, requiring only a small volume of sample and facilities available in many hospital laboratories. The method allows separate determination of the molar activities of cholic acid and chenodeoxycholic acid in the same sample, even when these bile acids are labelled with the same isotope. Material and methods Samples Duodenal aspirates were obtained after stimulation of the gallbladder emptying with 40-80 Ivy dog units of cholecystokinin (CCK-PZ, GIH Research Unit, Chemistry Department, Karolinska Institutet, Stockholm, Sweden) injected i.v. An aliquot of 0.5-1.5 ml of the volume aspirated at each time was retained for analysis. The aspirates were obtained at various intervals after i.v. administration of approximately 3.7 X 10’ Bq = 10 &i carboxy-i4C-cholate and 3.7 X 10’ Bq = 10 $!i carboxy-14C-chenodeoxycholate (The Radiochemical Centre, Amersham, U.K.). Choloylglycine hydrolase (EC 3.5.1.24) solution A solution of 15 mg of Clostridium welchii acetone powder, Type III (Sigma No. C-8635) per 1 ml of disodium hydrogen phosphate buffer, 0.2 mol/l, pH 5.8, was prepared. Thorough mixing for 5 min was followed by centrifugation at 2000 X g for 20 min. The supernatant contains the enzyme, capable of hydrolyzing glycine as well as taurine conjugates of bile acids. do-Hydroxysteroid dehydrogenase (3a-HSD) (EC 1 .l. 1.50) Sterognost-3cy@ from Nyegaard and Co. A/S, Oslo, Norway. Liquid scintillation counting solution Naphthalene 60 g, PPO (2,5-diphenyl-1,3-oxazole) 4 g, POPOP (1,4-bis( 2-( 5phenyl-oxazolyl))-benzene) 0.2 g, methanol 100 ml, ethylene glycol 20 ml, p-dioxane ad 1000 ml. Procedure Initially, the total bile acid concentration mined enzymatically (3ol-HSD) as previously
in the duodenal described [ 71.
sample was deter-
249
An appropriate volume (usually 100-300 ~1) of the sample, containing about 4 pmol of total bile acid, was subjected to enzymatic hydrolysis by mixing with 4 ml of the choloylglycine hydrolase solution and incubation of the mixture for 30 min in a water-bath at 37°C under constant shaking. After cooling to room temperature the pH of the mixture was adjusted to approximately 1 (pH-indicator paper) with 6 mol/l HCl. The deconjugated bile acids were then extracted three times with 2 ml of diethyl ether. The pooled ether extracts were evaporated to dryness at room temperature under a stream of nitrogen. The residue was redissolved in 100 ~1 of methanol and applied to a thin-layer plate with silica gel [ $1 in a 30 mm broad band, after which the chromatogram was developed according to previous description [ 81. In this way a satisfactory separation of the deconjugated bile acids, cholic acid, chenodeoxycholic acid, and deoxycholic acid, was obtained, although the latter two were situated rather near to each other. With the aid of reference solutions run in parallel on the same plate, the spots were detected by exposing the plate to iodine vapor and delineated. Thereafter, the iodine was allowed to sublimate at room temperature. The delineated bands with the individual bile acids were scraped off, in the case of chenodeoxycholic acid taking precautions to avoid the verges of the band. The removed silica gel was transferred to a glass-sintered column, and the bile acid was eluted with 15 ml of methanol. The methanol solution was evaporated to dryness on a water-bath at 7080°C under a stream of nitrogen, and the residue was redissolved in 2 ml of methanol. Of this volume, 1 ml (+ 10 ml of the counting solution) was taken for measurement of radioactivity in a Beckman liquid scintillation counter (LS 250), using external standardization to correct for quenching, and the remainder was used for determination of the concentration of bile acids by the 3a-HSD method (in duplicate) [ 71. The molar activity was calculated from the values obtained for activity concentration (Bq/l) and the substance concentration of bile acid (mol/l), the unit thus being Bq/mol. (The factor of conversion from the hitherto most commonly used unit “disintegrations per min/pmol” to Bq/mol is 1.67 X 104). Comparative method For comparison with the procedure described above, the molar activity of cholic acid was determined from the glycine and taurine conjugates of cholic acid. The sample was handled as previously stated [ 81, resulting in a chromatogram with separation of glycocholic acid and taurocholic acid from the dihydroxy bile acid conjugates. The removed silica gel spots with glycocholic acid and taurocholic acid, respectively, were pooled and thereafter followed the last part of the procedure described above.
250
Results and discussion The a~uly~~ca~ uariutio~ of the method was evaluated from the standard deviation for two totally independent results of duplicate analyses on different days of the same duodenal sample on 21 occasions (samples from five patients), the standard deviation being calculated from: d(EdF/2n). For cholic acid it was 6.6 X lo6 Bq/mol (interval of individual mean values 8.7 X 106-2.8 X 108, mean 1.1 X lo”), and for chenodeoxycholic acid 6.8 X lo6 Bq/mol (interval of individu~ mean values 2.6 X 107-1.9 X 108, mean 1.1 X 10X). Cholic acid was well separated from the other major bile acids, but as the spots of chenodeoxycholic acid and deoxycholic acid were situated rather near to each other, contamination of the removed chenodeoxycholic acid silica gel spot from the deoxycholic acid spot was a possibility, in spite of careful seraping of the central part of the spot. Rowever, the almost identical standard deviations {at the same level of molar activity) for cholic acid and chenodeoxycholic acid make it unlikely that significant contamination had occurred, as the possible contamination is unlikely to be of equal magnitude for the two scrapings of chenodeoxycholic acid for each result. Thus, it appears that the molar activity of both the major primary bile acids can be determined separately, even when labelled with the same isotope. The detection limit was taken as three times the standard deviation stated above, i.e. 19.8 X lo6 Bq/mol for cholic acid, and 20.4 X lo6 Bq/mol for chenodeoxycholic acid. The volume o~sarn~~e required for the analysis with the procedure described is small, which may be of decisive importance. If the Lindstedt technique [l] is used, involving serial determinations to obtain the molar activity elimination curve for the bile acids, the volume of duodenal sample (and thereby the amount of bile acids) retained for analysis should be as small as possible. In some of the current methods, the volume of sample retained is rather high, up to 10 ml of concentrated bile [5]. As the concentration of bile acids in duodenal aspirates after stimulation of the gallbladder emptying with cholecystokinin is usually 15-50 pmol/ml, a considerable amount of the bile acid pool (and isotope) is removed, significantly influencing the physiological steady state of the bile acid metabolism. For the ~~~~o~~sis of the conjugated bile acids an enzymatic method was used, which is in accordance with the recommendation to replace alkaline hydrolysis with an enzymatic procedure [9,10]. Furthermore, the enzymatic method is simpler. Our experiments showed that with 1 ml of the enzyme solution per 1 pmol of bile acid complete hydrolysis of glycine as well as of the taurine conjugates is achieved. A further advantage of the present method is that only the last steps in the procedure, the determination of radioactivity and of the bile acid concentration, need to be quantitative, and that the result of the analysis may be available in about 5 h .
251
Comparison of two methods Comparison of the values for the molar activity of cholic acid in duodenal samples with the two methods described was performed in 45 instances (4-5 samples from each of 10 patients). Theoretically, the values found with the two methods should be identical, i.e. that the ratio of the two results in each pair was 1.00, or, in practice, that the ratios would be evenly distributed about 1.00. However, the Wilcoxon test for pair differences showed significantly higher results with the hydrolytic procedure than with the other method @ < 0.01). The ratios of the values obtained with the deconjugated cholic acid to the values derived from the glycine and taurine conjugated cholic acid were in the interval of 0.93 to 1.27, median value 1.07, and only 6 out of the 45 ratios were below 1.00. It is difficult to ascertain which of the two results is the true one. A reasonable explanation for the difference is that some 3cu-hydroxy bile acid other than glycocholic acid and taurocholic acid and with a lower molar activity (possibly zero) than that of these was scraped off from the plate together with glycocholic acid and taurocholic acid. This would increase the bile acid concentration in that eluate to a higher degree than the radioactivity concentration, if the contaminating bile acid after hydrolysis does not appear in the spot of the unconjugated cholic acid. References 1 2 3 4 5 6 7 8 9 10
Lindstedt, S. (1957) Acta Phys. Stand. 40. l-9 Pomare, E.W. and Low-Beer, T.S. (1974) CIin. Chim. Acta 57. 239-248 Duane, W.C., Adler. R.D.. Bennion. L.J. and Ginsberg, R.L. (1975) J. Lipid Res. 16. 155-158 Vlahcevic, Z.R.. Be& C.C. Jr., Buhac, I., Farrar. J.T. and Swell. L. (1970) Gastroenterology 59,165173 Einarsson, K., HeIIstrSm. K. and KaIIner, M. (1974) J. CIin. Invest. 54,1301-1311 Hepner, G.W. (1975) Gastroenterology 68,1574-1581 Bruusgaard, A., Sdrensen. H.. Gilhuus-Moe, C.-C. and Sk&egg. B.A. (1977) CIin. Chim. Acta 77. 387395 Bruusgaard. A. (1970) CIin. Chim. Acta 28.495-504 Roovers, J., Evrard, E. and Vanderhaeghe. H. (1968) CIin. Chim. Acta 19. 449457 Eneroth, P. and Sj6vaB. J. (1971) in The Bile Acids, Volume 1: Chemistry (Nair, P.P. and Kritchevsky, D., eds.). p. 133, Plenum Press, New York -London
