127
Clinica Chimica Acta, 64 (1975) 127-132 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7218
DETERMINATION
MARION
J. SHELTAWY
University
Department
(Received
May 5, 1975)
OF FAECAL BILE ACIDS BY AN ENZYMIC METHOD
and M.S. LOSOWSKY of Medicine,
St. James’s
Hospital,
Leeds
LS9
7TF
(U.K.)
Summary A relatively simple, cheap reliable method for determining faecal bile acids, using 3cr-hydroxy steroid dehydrogenase is presented. Good recoveries were obtained even in the presence of very high fat concentrations. The mean value for a group of patients without gastrointestinal disease is reported.
Introduction The determination of faecal bile acids provides an accurate measure of the daily synthesis of bile acids from cholesterol by the liver [ 11, and it can also contribute to the diagnosis of diseases of the ileum in which bile salt malabsorption is a feature. However, the measurement of faecal bile acids is complicated by the wide range of polarity of the bacterial degradation products of the two primary bile acids, cholic and chenodeoxycholic acid [2,3] . Although methods involving gas-liquid chromatography have been validated [3,4], they involve extensive initial purification and require considerable expertise. Several enzymatic methods have been described [ 5,6] , but, although requiring less initial purification, are impractical for most laboratories as they use large volumes of solvent and multi-stage procedures. In the method presented the procedures have been scaled down, simplified and sources of error eliminated. Materials 3cu-Hydroxy steroid dehydrogenase was obtained from Sigma Chemical Company, Surbiton, Surrey, as a soluble powder (Grade II) from Pseudomonas testosteronii containing both 3a- and 3&enzymes. This was dissolved in water to give a solution containing 0.5 units/ml and was stored froz&. NAD was obtained from Sigma Chemical Company, London. 3-(Cyclohexylamino)propane sulphonic acid (CAPS) was obtained from Hopkins and Williams, Essex, and made up as a 0.02 M solution, pH 10.8. Girard T, hydrazine
128
hydrate and semicarbazide hydrochloride were obtained from British Drug Houses, Poole, Dorset. Once a fortnight a 0.5 M solution was prepared in 0.1 M pyrophosphate buffer and adjusted to pH 10.0. Girard T was found to be unstable in CAPS buffer. Bile salts were obtained from Koch-Light, Colnbrook, Bucks. Lithocholate was purified by precipitation from ethanol by 0.1 N HCl. Solvents were analytical reagent grade. Methanol was redistilled over 2,4-dinitrophenyl hydrazine. Silica Gel G was obtained from E. Merck, Darmstadt. Methods Extraction
of bile acids
Forty-eight hour stool collections contained chromium sesquioxide and polyethylene glycol 4000 as continuous, non-absorbable markers. Homogenisation was carried out by adding warm water to the specimen in a Kenwood mixer. Three to five minutes of high speed homogenisation was sufficient to reduce any fibrous matter to fine particles. The final consistency was a smooth running paste from which the chromium sesquioxide did not readily settle. An aliquot (0.2-0.5 g) of the homogenate was weighed into a 50 ml test tube with clearfit joints. Six ml 2 N NaOH were added and a fine piece of stainless steel wire inserted between the tube and stopper to permit steam to escape. The samples were then autoclaved so that they had a full 90 min under a pressure of 16 psi at 120°C. At the end of this period, preferably while the tubes were still warm, 20 ml ethyl acetate was added and the tubes were shaken manually for 30 s. Then 2 ml cont. HCl were added, mixed by gentle agitation, and after cooling for a few minutes, the tubes were securely stoppered and shaken for a further 10 min on a mechanical shaker. They were then centrifuged and the upper layer transferred to a 50 ml tube. A second extraction using 10 ml ethyl acetate was performed and the combined upper phases evaporated to dryness at 65°C under a stream of nitrogen. Removal
of lipids
The residue was taken up carefully in 0.2-0.5 ml propanol and transferred quantitatively to a thin-layer plate of Silica gel G (thickness 0.3 mm) to form a narrow band (7 cm X 1 cm). The plate was developed in ether/hexane/glacial acetic acid (30 : 70 : 1, v/v). Standards of lithocholate, fatty acid and cholesterol were run at the edges of the plate, and after development were sprayed with 0.05% solution of 2’,7’-dichloro-fluorescein in 90% ethanol, while the areas containing faecal bile acids were masked. The plate was viewed under ultraviolet light and the areas including lithocholate and the origin scraped off. This permitted removal of the cholesterol and other sterols which run well ahead of lithocholate. The silica gel containing the bile acids was extracted for 15 min on a mechanical shaker, first with 10 ml and then with 5 ml of 2.5% acetic acid in methanol. The supernatants were combined and evaporated to dryness. Since it is easy to lose some lithocholate at this stage, the bile salt residue was warmed and carefully extracted with small aliquots of methanol, and these were transferred to a 1.0 ml volumetric flask. If particles were present in the methanolic extract, these were dispersed before aliquoting.
129
Enzyme
assay
The incubation reagent, prepared immediately before use, consisted of 0.5 M Girard T solution or 0.5 M hydrazine hydrate or 0.5 M semicarbazide HCl (ketone trapping agents) and 0.02 M CAPS buffer (1 : 19, v/v) to which was added 5 mg NAD/BO ml buffer mixture. To a stoppered cuvette was added 2 ml incubation reagent and 0.1 ml of methanolic solution of bile salt, the contents were mixed and placed to warm at 37°C for 10 min. The absorbance 340 nm was read and 0.1 ml hydroxy steroid dehydrogenase (0.05 units) was added immediately and mixed. Absorbance readings were then taken every 5 min with the cuvettes maintained at 37°C. The reaction was complete by 10 min but the absorbance continued to rise slowly due to other reactions. The slope was extrapolated back to zero time and the absorbance increment thus calculated was a measure of the amount of bile salts present. This value was read off a calibration curve constructed with cholic acid standards. A lithocholate standard of 300 pg/ml was included in each run since this is the most slowly oxidised bile acid and the speed of the reaction can indicate exhaustion of reagents. Care with washing up is essential, traces of detergent cause substantial error and cuvettes are best soaked immediately after use for about 1 h in warm, dilute KOH, and then rinsed exhaustively in tap and distilled water. Results From pH 9.5 to 10.8 the oxidation of the 3a-hydroxyl group was complete within ten minutes when the bile acid was cholic or deoxycholic acid. The reaction with lithocholate was however, slower, but its rate increased with increasing pH (Fig. 1). The oxidation of lithocholate was also more sensitive to exhaustion of reagents than that of other bile salts. The calibration curve of cholic acid, deoxycholic acid and lithocholate acid was linear up to 140 nmol per assay. The three bile acids gave identical optical density readings for equimolar amounts (Fig. 2). The same calibration A340
“In
o-4 ../
1
./ A340 0.3
“In
-.mi-.-.
.’
0,3 7-.=.-
-
./’
-
/I
-
0.2
,,/
0.2 :/’ i
1 0.1
0.1 /
5
IO
15
20 TIME
25
30
35
40
40
80 .
(MINS)
Fig. 1. Rate of reaction of lithocholate acid (80 nmol) at pH 9.5 (ON PH 10.8 (A). Each value is the mean of four estimations.
), pH 10.2
Fig. 2. Calibration curve for cholic acid (0). deoxycholic acid (A), lithocholate salt is expressed as nmole per assay. Each value is the mean of four estimations.
120
160
BILESALThmomoks) (0-j
acid (0).
Amount
and at
of bile
130 TABLE
I
RECOVERY
OF
Weight
(/.nnol)
added
BILE
SALTS
FROM
Bile
0.2
salt
g FATTY
FAECES
Mean
percentage
recovery
Standard
0.73
Deoxycholate
98.2
2.36
0.80
Lithocholate
96.4
1.54
0.56
Tawocholate
86.4
0.99
0.61
Glycocholate
88.8
2.64
error
curve was obtained for each ketone trapping agent tested, but semicarbazide was the most satisfactory and was selected for routine use. Recovery experiments were performed by adding known amounts of standards to faecal homogenate from a patient with complete obstructive jaundice, containing about 75 mg fat per gram and no bile acids. This represents as high a level of faecal fat as is likely to be encountered. The recoveries obtained are shown in Table I. In the case of glycine and taurine conjugates of cholic acid some bile acid was lost almost certainly due to incomplete hydrolysis or partial destruction of the cholate molecule. Recovery of deoxycholate and lithocholate was nearly complete when care was taken to redissolve all the bile salts after evaporation to dryness. Addition of [ 14C] cholic acid to the initial sample in order to correct for losses was not considered helpful since each bile acid behaves differently and is subject to losses at different stages of the procedure. That no compounds other than bile salts were estimated was shown by zero values in a faecal sample from a patient with total obstruction of the common bile duct. Using this method, the range of daily bile salt excretion found in a group of eleven patients without gastrointestinal or liver disease, and without constipation or diarrhoea was 245-880 pmol (100-360 mg) with a mean of 490 pmol (202 mg). The mean value based on body weight was 8.0 rt 0.8 pmol/kg body weight (3.3 * 0.3 mg). As an index of the overall precision of the method, the standard deviation (S.D.) between duplicates for a series of samples can be calculated using the formula S.D. = d(Z(X, -X,)2)/2 N. The SD. between duplicates for the control series of patients mentioned above was 36 pmol (14.7 mg). For a second series of twelve patients excreting lo-40 g fat per day and 196-760 pmol (80--3lO.mg) bile salts per day, the S.D. was 25.8 pmol (10.5 mg) per day. Discussion The measurement of faecal bile acids involves multistage procedures because of the range of bile acid derivatives present and because of the interfering pigments and colonic bacterial degradation products not normally present in other biological fluids. The availability of an enzyme specific for the Sa-hydroxyl group of C-24 steroids which is possessed by about 90% of faecal bile acids [7], promises to
131
provide an easier method than gas-liquid chromatography, but existing methods using this enzyme [5,6] have a number of shortcomings. The use of large samples of faecal homogenate necessitates large volumes of solvents for extraction, which are costly and unmanageable in ordinary laboratory equipment. We weighed small amounts of a well mixed homogenate and used much smaller volumes of solvents. An initial stage of extraction of neutral lipids before autoclaving which has been included by other workers [4,6] can lead to losses of lithocholate [ 71. We found it unnecessary as subsequent thin-layer chromatography removed both fatty acid and sterols. Development in a more polar solvent than that commonly used [ 51 enabled complete separation of bile salts and lipids without loss of resolution. Removal of the bile salts quantitatively from the silica gel required vigorous mechanical shaking. Many workers [4,6] use ethanolic KOH or NaOH to hydrolyse the peptide bond of conjugated bile salts during autoclaving. However, ethanolic NaOH solutions rapidly develop coloured impurities and also foam during autoclaving. We have found aqueous 2 N NaOH more satisfactory, enabling the use of 50 ml “clearfit” tubes in which are carried out subsequent shaking and centrifugation steps. A source of error not previously described is the tendency of lithocholate to precipitate out on acidification and bind tenaciously to debris, glass and the chromic oxide present as a faecal marker. We found this loss was eliminated by a brief extraction into ethyl acetate before as well as after acidification. The final assay was modified in a number of ways. It was found that lithocholate was slower to react with the enzyme than other bile salts and was particularly sensitive to exhaustion of reagents. This effect was less pronounced at pH 10.8, and as pyrophosphate does not buffer above pH 9.8, the zwitterionic buffer, CAPS (pK, 10.4) was substituted and found very satisfactory. CAPS is itself a detergent, and lowered the surface tension so that air bubbles were not trapped on the optical surface of the cuvette. 3a-Hydroxy steroiddehydrogenase is contaminated with a broad specificity, NAD-linked alcohol dehydrogenase which catalyses the oxidation of hydroxyl groups on a variety of molecules to give a very slow but continuous rise in the absorbance reading over one to two hours. Traces of propanol, butanol, ethanediol, glycerol and ethanol all produced this effect. Since, non-volatile compounds of this type may be produced during autoclaving, it is necessary to record the increment due to these other reactions by taking absorbance readings at time intervals so that it can be subtracted from that due to oxidation of the 3a-hydroxyl group of the bile salts. Skalhegg [8] , has reported that some preparations of 3a-hydroxyl steroid dehydrogenase also contain an NAD-linked 12a-hydroxy steroid dehydrogenase. This is not a contaminant of the preparation we have used since equimolar amounts of the three bile salts gave the same absorbance rise. The normal range of daily bile salt excretion is known to depend on diet [9] particularly its fibre content [lo] and also bowel habit, in that the slower the transit time the greater the reabsorption of bile salts from the colon. The normal range obtained by this method compares favourably with that obtained
132
using gas-liquid chromatography by Evrard and Janssen [3], 127-290 mg per day (mean 218 mg per day) by Miettinen [l], 238 t 25 mg per day or 3.8 t 0.23 mg/kg per day and by Ali and his colleagues [7], 130-165 mg per day. The method described is relatively simple, cheap and reliable and requires less expertise and special equipment than methods employing gas-liquid chromatography. It is therefore more suitable for laboratories which do not specialise in bile salt analysis. Acknowledgements This work was supported by a grant from the Medical Research Council. We are also grateful to Mrs Susan E. Melling for excellent technical assistance. References 1
T.A.
Miettinen,
lism,
p. 196,
2
P. Eneroth.
3
E. Evrard
4
S.M.
Grundy,
5
J.F.
Woodbury
A.M. S.S.
Weber,
8
B.A.
Skalhegg,
9
B.S.
Reddy
H.J.
Birkner
A.
and
Stand.
and and
and
F. Kern.
L. Chartrand, Kukiss
E.L.
D. Kritchevsky York,
J. Lipid
Ahrens
and
and New
R. Ryhage
G. Janssen, E.I..
7
10
Nair
Press,
B. Gordon, and
6
Ali,
in P.P. Plenum
and J. Sjovall.
J. Lipid 226
T.A.
G. Doyen,
9 (1968)
Miettinen. Invest.,
F. Kern,
J. Natl.
J. Lipid 50
(1971)
S. Gordon
Beveridge,
J. Gastroenterol.. Wynder,
The
Res..
J. Clin.
J.M.R.
(eds),
Bile
Acids,
Vol.
2. Physiology
and
Metabo-
1973
Can.
and
Gastroenterology,
Res.,
6 (1965)
C.C.
Roy, 44
50 (1973)
(1974)
511 397
Clin.
Chin
(1966)
957
555
Inst., 67
7 (1966)
2531
J. Biochem..
9 (1974) Cancer
Res.,
237
1437
Acta
39
(1972)
524
Clinica Chimica Acta, 64 (1975) 127-132 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7218
DETERMINATION
MARION
J. SHELTAWY
University
Department
(Received
May 5, 1975)
OF FAECAL BILE ACIDS BY AN ENZYMIC METHOD
and M.S. LOSOWSKY of Medicine,
St. James’s
Hospital,
Leeds
LS9
7TF
(U.K.)
Summary A relatively simple, cheap reliable method for determining faecal bile acids, using 3cr-hydroxy steroid dehydrogenase is presented. Good recoveries were obtained even in the presence of very high fat concentrations. The mean value for a group of patients without gastrointestinal disease is reported.
Introduction The determination of faecal bile acids provides an accurate measure of the daily synthesis of bile acids from cholesterol by the liver [ 11, and it can also contribute to the diagnosis of diseases of the ileum in which bile salt malabsorption is a feature. However, the measurement of faecal bile acids is complicated by the wide range of polarity of the bacterial degradation products of the two primary bile acids, cholic and chenodeoxycholic acid [2,3] . Although methods involving gas-liquid chromatography have been validated [3,4], they involve extensive initial purification and require considerable expertise. Several enzymatic methods have been described [ 5,6] , but, although requiring less initial purification, are impractical for most laboratories as they use large volumes of solvent and multi-stage procedures. In the method presented the procedures have been scaled down, simplified and sources of error eliminated. Materials 3cu-Hydroxy steroid dehydrogenase was obtained from Sigma Chemical Company, Surbiton, Surrey, as a soluble powder (Grade II) from Pseudomonas testosteronii containing both 3a- and 3&enzymes. This was dissolved in water to give a solution containing 0.5 units/ml and was stored froz&. NAD was obtained from Sigma Chemical Company, London. 3-(Cyclohexylamino)propane sulphonic acid (CAPS) was obtained from Hopkins and Williams, Essex, and made up as a 0.02 M solution, pH 10.8. Girard T, hydrazine
128
hydrate and semicarbazide hydrochloride were obtained from British Drug Houses, Poole, Dorset. Once a fortnight a 0.5 M solution was prepared in 0.1 M pyrophosphate buffer and adjusted to pH 10.0. Girard T was found to be unstable in CAPS buffer. Bile salts were obtained from Koch-Light, Colnbrook, Bucks. Lithocholate was purified by precipitation from ethanol by 0.1 N HCl. Solvents were analytical reagent grade. Methanol was redistilled over 2,4-dinitrophenyl hydrazine. Silica Gel G was obtained from E. Merck, Darmstadt. Methods Extraction
of bile acids
Forty-eight hour stool collections contained chromium sesquioxide and polyethylene glycol 4000 as continuous, non-absorbable markers. Homogenisation was carried out by adding warm water to the specimen in a Kenwood mixer. Three to five minutes of high speed homogenisation was sufficient to reduce any fibrous matter to fine particles. The final consistency was a smooth running paste from which the chromium sesquioxide did not readily settle. An aliquot (0.2-0.5 g) of the homogenate was weighed into a 50 ml test tube with clearfit joints. Six ml 2 N NaOH were added and a fine piece of stainless steel wire inserted between the tube and stopper to permit steam to escape. The samples were then autoclaved so that they had a full 90 min under a pressure of 16 psi at 120°C. At the end of this period, preferably while the tubes were still warm, 20 ml ethyl acetate was added and the tubes were shaken manually for 30 s. Then 2 ml cont. HCl were added, mixed by gentle agitation, and after cooling for a few minutes, the tubes were securely stoppered and shaken for a further 10 min on a mechanical shaker. They were then centrifuged and the upper layer transferred to a 50 ml tube. A second extraction using 10 ml ethyl acetate was performed and the combined upper phases evaporated to dryness at 65°C under a stream of nitrogen. Removal
of lipids
The residue was taken up carefully in 0.2-0.5 ml propanol and transferred quantitatively to a thin-layer plate of Silica gel G (thickness 0.3 mm) to form a narrow band (7 cm X 1 cm). The plate was developed in ether/hexane/glacial acetic acid (30 : 70 : 1, v/v). Standards of lithocholate, fatty acid and cholesterol were run at the edges of the plate, and after development were sprayed with 0.05% solution of 2’,7’-dichloro-fluorescein in 90% ethanol, while the areas containing faecal bile acids were masked. The plate was viewed under ultraviolet light and the areas including lithocholate and the origin scraped off. This permitted removal of the cholesterol and other sterols which run well ahead of lithocholate. The silica gel containing the bile acids was extracted for 15 min on a mechanical shaker, first with 10 ml and then with 5 ml of 2.5% acetic acid in methanol. The supernatants were combined and evaporated to dryness. Since it is easy to lose some lithocholate at this stage, the bile salt residue was warmed and carefully extracted with small aliquots of methanol, and these were transferred to a 1.0 ml volumetric flask. If particles were present in the methanolic extract, these were dispersed before aliquoting.
129
Enzyme
assay
The incubation reagent, prepared immediately before use, consisted of 0.5 M Girard T solution or 0.5 M hydrazine hydrate or 0.5 M semicarbazide HCl (ketone trapping agents) and 0.02 M CAPS buffer (1 : 19, v/v) to which was added 5 mg NAD/BO ml buffer mixture. To a stoppered cuvette was added 2 ml incubation reagent and 0.1 ml of methanolic solution of bile salt, the contents were mixed and placed to warm at 37°C for 10 min. The absorbance 340 nm was read and 0.1 ml hydroxy steroid dehydrogenase (0.05 units) was added immediately and mixed. Absorbance readings were then taken every 5 min with the cuvettes maintained at 37°C. The reaction was complete by 10 min but the absorbance continued to rise slowly due to other reactions. The slope was extrapolated back to zero time and the absorbance increment thus calculated was a measure of the amount of bile salts present. This value was read off a calibration curve constructed with cholic acid standards. A lithocholate standard of 300 pg/ml was included in each run since this is the most slowly oxidised bile acid and the speed of the reaction can indicate exhaustion of reagents. Care with washing up is essential, traces of detergent cause substantial error and cuvettes are best soaked immediately after use for about 1 h in warm, dilute KOH, and then rinsed exhaustively in tap and distilled water. Results From pH 9.5 to 10.8 the oxidation of the 3a-hydroxyl group was complete within ten minutes when the bile acid was cholic or deoxycholic acid. The reaction with lithocholate was however, slower, but its rate increased with increasing pH (Fig. 1). The oxidation of lithocholate was also more sensitive to exhaustion of reagents than that of other bile salts. The calibration curve of cholic acid, deoxycholic acid and lithocholate acid was linear up to 140 nmol per assay. The three bile acids gave identical optical density readings for equimolar amounts (Fig. 2). The same calibration A340
“In
o-4 ../
1
./ A340 0.3
“In
-.mi-.-.
.’
0,3 7-.=.-
-
./’
-
/I
-
0.2
,,/
0.2 :/’ i
1 0.1
0.1 /
5
IO
15
20 TIME
25
30
35
40
40
80 .
(MINS)
Fig. 1. Rate of reaction of lithocholate acid (80 nmol) at pH 9.5 (ON PH 10.8 (A). Each value is the mean of four estimations.
), pH 10.2
Fig. 2. Calibration curve for cholic acid (0). deoxycholic acid (A), lithocholate salt is expressed as nmole per assay. Each value is the mean of four estimations.
120
160
BILESALThmomoks) (0-j
acid (0).
Amount
and at
of bile
130 TABLE
I
RECOVERY
OF
Weight
(/.nnol)
added
BILE
SALTS
FROM
Bile
0.2
salt
g FATTY
FAECES
Mean
percentage
recovery
Standard
0.73
Deoxycholate
98.2
2.36
0.80
Lithocholate
96.4
1.54
0.56
Tawocholate
86.4
0.99
0.61
Glycocholate
88.8
2.64
error
curve was obtained for each ketone trapping agent tested, but semicarbazide was the most satisfactory and was selected for routine use. Recovery experiments were performed by adding known amounts of standards to faecal homogenate from a patient with complete obstructive jaundice, containing about 75 mg fat per gram and no bile acids. This represents as high a level of faecal fat as is likely to be encountered. The recoveries obtained are shown in Table I. In the case of glycine and taurine conjugates of cholic acid some bile acid was lost almost certainly due to incomplete hydrolysis or partial destruction of the cholate molecule. Recovery of deoxycholate and lithocholate was nearly complete when care was taken to redissolve all the bile salts after evaporation to dryness. Addition of [ 14C] cholic acid to the initial sample in order to correct for losses was not considered helpful since each bile acid behaves differently and is subject to losses at different stages of the procedure. That no compounds other than bile salts were estimated was shown by zero values in a faecal sample from a patient with total obstruction of the common bile duct. Using this method, the range of daily bile salt excretion found in a group of eleven patients without gastrointestinal or liver disease, and without constipation or diarrhoea was 245-880 pmol (100-360 mg) with a mean of 490 pmol (202 mg). The mean value based on body weight was 8.0 rt 0.8 pmol/kg body weight (3.3 * 0.3 mg). As an index of the overall precision of the method, the standard deviation (S.D.) between duplicates for a series of samples can be calculated using the formula S.D. = d(Z(X, -X,)2)/2 N. The SD. between duplicates for the control series of patients mentioned above was 36 pmol (14.7 mg). For a second series of twelve patients excreting lo-40 g fat per day and 196-760 pmol (80--3lO.mg) bile salts per day, the S.D. was 25.8 pmol (10.5 mg) per day. Discussion The measurement of faecal bile acids involves multistage procedures because of the range of bile acid derivatives present and because of the interfering pigments and colonic bacterial degradation products not normally present in other biological fluids. The availability of an enzyme specific for the Sa-hydroxyl group of C-24 steroids which is possessed by about 90% of faecal bile acids [7], promises to
131
provide an easier method than gas-liquid chromatography, but existing methods using this enzyme [5,6] have a number of shortcomings. The use of large samples of faecal homogenate necessitates large volumes of solvents for extraction, which are costly and unmanageable in ordinary laboratory equipment. We weighed small amounts of a well mixed homogenate and used much smaller volumes of solvents. An initial stage of extraction of neutral lipids before autoclaving which has been included by other workers [4,6] can lead to losses of lithocholate [ 71. We found it unnecessary as subsequent thin-layer chromatography removed both fatty acid and sterols. Development in a more polar solvent than that commonly used [ 51 enabled complete separation of bile salts and lipids without loss of resolution. Removal of the bile salts quantitatively from the silica gel required vigorous mechanical shaking. Many workers [4,6] use ethanolic KOH or NaOH to hydrolyse the peptide bond of conjugated bile salts during autoclaving. However, ethanolic NaOH solutions rapidly develop coloured impurities and also foam during autoclaving. We have found aqueous 2 N NaOH more satisfactory, enabling the use of 50 ml “clearfit” tubes in which are carried out subsequent shaking and centrifugation steps. A source of error not previously described is the tendency of lithocholate to precipitate out on acidification and bind tenaciously to debris, glass and the chromic oxide present as a faecal marker. We found this loss was eliminated by a brief extraction into ethyl acetate before as well as after acidification. The final assay was modified in a number of ways. It was found that lithocholate was slower to react with the enzyme than other bile salts and was particularly sensitive to exhaustion of reagents. This effect was less pronounced at pH 10.8, and as pyrophosphate does not buffer above pH 9.8, the zwitterionic buffer, CAPS (pK, 10.4) was substituted and found very satisfactory. CAPS is itself a detergent, and lowered the surface tension so that air bubbles were not trapped on the optical surface of the cuvette. 3a-Hydroxy steroiddehydrogenase is contaminated with a broad specificity, NAD-linked alcohol dehydrogenase which catalyses the oxidation of hydroxyl groups on a variety of molecules to give a very slow but continuous rise in the absorbance reading over one to two hours. Traces of propanol, butanol, ethanediol, glycerol and ethanol all produced this effect. Since, non-volatile compounds of this type may be produced during autoclaving, it is necessary to record the increment due to these other reactions by taking absorbance readings at time intervals so that it can be subtracted from that due to oxidation of the 3a-hydroxyl group of the bile salts. Skalhegg [8] , has reported that some preparations of 3a-hydroxyl steroid dehydrogenase also contain an NAD-linked 12a-hydroxy steroid dehydrogenase. This is not a contaminant of the preparation we have used since equimolar amounts of the three bile salts gave the same absorbance rise. The normal range of daily bile salt excretion is known to depend on diet [9] particularly its fibre content [lo] and also bowel habit, in that the slower the transit time the greater the reabsorption of bile salts from the colon. The normal range obtained by this method compares favourably with that obtained
132
using gas-liquid chromatography by Evrard and Janssen [3], 127-290 mg per day (mean 218 mg per day) by Miettinen [l], 238 t 25 mg per day or 3.8 t 0.23 mg/kg per day and by Ali and his colleagues [7], 130-165 mg per day. The method described is relatively simple, cheap and reliable and requires less expertise and special equipment than methods employing gas-liquid chromatography. It is therefore more suitable for laboratories which do not specialise in bile salt analysis. Acknowledgements This work was supported by a grant from the Medical Research Council. We are also grateful to Mrs Susan E. Melling for excellent technical assistance. References 1
T.A.
Miettinen,
lism,
p. 196,
2
P. Eneroth.
3
E. Evrard
4
S.M.
Grundy,
5
J.F.
Woodbury
A.M. S.S.
Weber,
8
B.A.
Skalhegg,
9
B.S.
Reddy
H.J.
Birkner
A.
and
Stand.
and and
and
F. Kern.
L. Chartrand, Kukiss
E.L.
D. Kritchevsky York,
J. Lipid
Ahrens
and
and New
R. Ryhage
G. Janssen, E.I..
7
10
Nair
Press,
B. Gordon, and
6
Ali,
in P.P. Plenum
and J. Sjovall.
J. Lipid 226
T.A.
G. Doyen,
9 (1968)
Miettinen. Invest.,
F. Kern,
J. Natl.
J. Lipid 50
(1971)
S. Gordon
Beveridge,
J. Gastroenterol.. Wynder,
The
Res..
J. Clin.
J.M.R.
(eds),
Bile
Acids,
Vol.
2. Physiology
and
Metabo-
1973
Can.
and
Gastroenterology,
Res.,
6 (1965)
C.C.
Roy, 44
50 (1973)
(1974)
511 397
Clin.
Chin
(1966)
957
555
Inst., 67
7 (1966)
2531
J. Biochem..
9 (1974) Cancer
Res.,
237
1437
Acta
39
(1972)
524
