Clinical Science and Molecular Medicine (1974) 46, 183-190.
THE FORMATION O F DEOXYCHOLIC ACID AND
CHENODEOXYCHOLIC ACID I N MAN
K U R T EINARSSON
AND
K J E L L HELLSTRUM
Department of Medicine, Serajimerlasarettet, Stockholm, Sweden
(Received 20 August 1973)
SUMMARY
1. The turnover of deoxycholic acid and chenodeoxycholic acid was studied in six
normolipaemic patients after oral administration of trace amounts of isotopically
labelled compounds.
2. The mean values for half-life, pool size and turnover of deoxycholic acid were
3.0 days, 663 mg and 171 mg/day respectively. The corresponding values recorded for
chenodeoxycholic acid were 2.8 days, 781 mg and 207 mg/day.
3. A comparison of the turnover rates of deoxycholic acid and cholic acid in
three subjects indicated that 25-61% of the cholic acid was converted into deoxycholic acid.
4. Only trace amounts of radioactivity were recovered in the trihydroxycholanic
acid fraction of duodenal bile after the administration of [14C]deoxycholic acid or
[3H]chenodeoxycholicacid.
Key words : deoxycholic acid, chenodeoxycholic acid, cholic acid, duodenal bile, turnover studies.
Deoxycholic acid is quantitatively the most important product derived from cholic acid during
its enterohepatic circulation. This metabolite is effectively absorbed from the intestine and
accounts for about 20% of the bile acids in human bile. By measuring the specific radioactivity
of cholic acid and deoxycholic acid in specimens of duodenal bile obtained after administration of labelled cholesterol it appears that 10-90% of the cholic acid formed is converted into
deoxycholic acid (Lindstedt, 1970). This bile acid is normally present in faeces together with
other cholic acid metabolites such as 3~-hydroxy-12-oxo-5~-cholanic
acid, 12-oxolithocholic
acid and 3P,12a-dihydroxy-5&cholanic acid (Danielsson, Eneroth, Hellstrom, Lindstedt &
Sjovall, 1963). The metabolic interrelations between these bile acids have not been established.
Owing to technical difficulties, it has not been possible to perform a simultaneous study of
Correspondence: Dr Kjell Hellstrom, Department of Medicine, Serafimerlasarettet, S-112 83 Stockholm,
Sweden.
183
Kurt Einarsson and Kjell Hellstrom
184
the kinetics of the three major bile acids found in human bile. Most interest has been focused on
determinations of the total bile acid formation, i.e. the turnover of cholic acid and chenodeoxycholic acid. However, recent observations concerning the mechanism regulating the bile acid
formation in intact animals (Dowling, Mack & Small, 1970) underline the importance of
knowing the size of the total bile acid pool and the number of enterohepatic circulations it
undergoes. The aim of this investigation was to study the turnover of deoxycholic acid in
normolipaemic subjects.
EXPERIMENTAL
Patients
The patients who volunteered for the investigation were 25-66-year-old men (Table 1).
Patient 2 had undergone cholecystectomy and partial thyroidectomy because of a benign
TAEILE
1. Details of patients studied
Serum
Serum
cholesterol(l) triglycerides")
(mg/100 ml)
(mg/100 ml)
Patient
no.
Age
(years)
Height
(cm)
Weight
1 (K.J.)
2 (S.O.)
54
45
167
167
68
67
267
21 8
139
147
3 (L.M.)
4 (V.A.)
25
48
177
176
81
86
153
218
155
160
5 (G.N.)
57
164
75
220
185
6 (S.H.)
66
176
73
272
135
14&285
80-1 80
(kg)
Normal range
(l)
Previous history and
present symptoms
Angina pectoris, spondylosis
Partial thyroidectomy,
cholecystectomy, nephrolithiasis
Nephrolithiasis
Myocardial infarction, angina
pectoris
Myocardial infarction,
angina pectoris
Myocardial infarction,
angina pectoris
Mean of several determinations.
adenoma. He received thyroid hormone replacement therapy. Four patients had coronary
heart disease and patient 3 had nephrolithiasis. Patients 4 and 5 were moderately overweight;
when re-examined after 6-9 months, they had lost about 5 kg in weight.
The patients were normolipaemic as judged by repeated measurements of serum cholesterol
and triglyceride and by the pattern of distribution of the lipoproteins upon electrophoresis on
agarose gel.
Experimental procedure
The patients were given a standardized diet of natural food for some days before and during
the experimental period. The total energy intake was approximately 7.14 kJ (1.700 kcal)/day
and 35% of the energy was supplied as fat. The daily intake of cholesterol was about 300 mg.
The sodium salts of [14C]deoxycholicacid (4 pCi) and [3H]chenodeoxycholic acid (15 pCi)
Turnover of bile acids
185
were dissolved in water and administered orally before breakfast. During a second study, 6-9
months later, patients 4-6 received a mixture of ['4C]cholic acid (4 pCi) and [3H]chenodeoxycholic acid (15 pCi). On four occasions at 2-3 day intervals a polyvinyl tube was placed in the
duodenum and a sample of about 10 ml of concentrated bile was obtained after an intravenous
injection of cholecystokinin. Serum samples were obtained twice a week and analysed for
cholesterol, triglyceride and lipoprotein pattern.
Materials
[24-14C]Cholicacid (138 pCi/mg) was purchased from New England Nuclear Corp., Boston,
Mass., U.S.A. [24-14C]Deoxycholic acid (18.8 pCi/mg) was manufactured by Mallinckrodt
Nuclear, St Louis, Mo., U.S.A. The radiochemicalpurity (>99%) of the isotopes was checked
by radioautography on thin-layer chromatograms. Randomly labelled [3H]chenodeoxycholic
acid (40 pCi/mg) was a gift from Dr H. Danielsson, Stockholm. It was prepared by the method
of Wilzbach (1957), purified by various chromatographic procedures and finally recrystallized
to constant specific radioactivity. Cholecystokinin was obtained from the Gastrointestinal
Hormone Group, Chemical Department, Karolinska Institutet, Stockholm, Sweden.
Methods
The bile samples were hydrolysed with KOH (1 mol/l) in closed steel tubes for 12 h at
110°C. After acidification to pH 1-2, the bile acids were extracted with diethyl ether. The
ether extracts were washed with water until neutral and evaporated to dryness. The residue was
subjected to reverse-phasepartition chromatography. Hostalene (Farbwerke Hoechst G.m.b.H.,
West Germany) was used as the supporting material, methanol-water (165: 135, v/v) as the
moving phase and chloroform-heptane (45:5, v/v) as the stationary phase (Norman &
Sjovall, 1958). Two titration peaks were obtained, corresponding to the trihydroxycholanic
acids and the dihydroxycholanic acids. Cholic acid was recrystallized from ethyl acetate and
assessed for I4C radioactivity. After evaporation to dryness one aliquot of the dihydroxycholanic acid fraction was dissolved in methanol-ether (1 :9, v/v), methylated with diazomethane, treated with trifluoroacetic anhydride and analysed by gas-liquid chromatography
(Sjovall, 1962). Another aliquot was analysed for radioactivity and the specific radioactivities
of the two acids were calculated, assuming that all the I4C and 3H in the dihydroxycholanic
acid fraction was present as deoxycholic acid and chenodeoxycholic acid respectively (Danielsson et al., 1963).
The half-life, pool size and turnover of the bile acids and the concentrations of serum lipids
were determined as described in a previous paper (Einarsson & Hellstrom, 1972).
RESULTS
The half-lives of deoxycholic acid and chenodeoxycholic acid ranged between 1.3 and 4.1 days
(Table 2). Relatively large individual variations were observed for the pool sizes, which averaged 663 (range 372-1108) mg for deoxycholic acid and 781 (363-1120) mg for chenodeoxycholic acid. The ratios of the pool sizes of the two acids did not correlate well with the ratios of
the concentrations of the compounds in duodenal aspirates. This discrepancy was due mainly
to the relatively large variation (up to 100%) in the values for the ratio of chenodeoxycholic
acid to deoxycholic acid concentrations in the bile (Fig. 1). The mean turnover of deoxycholic
Kurt Einarsson and Kjell Hellstrom
186
TABLE
2. Turnover of deoxycholic acid and chenodeoxycholic acid
Deoxycholic acid
Patient
no.
1
2
3
4
5
6
MeankSD
Chenodeoxycholic acid
Half-life
(days)
Pool size
(mg)
Turnover
(mg/dw)
Half-life
(days)
Pool size
(mg)
Turnover
(mg/day)
2.9
4.0
2.8
3.3
3.0
1.7
552
664
1108
562
372
719
132
115
275
118
86
300
4.0
1.8
4.1
2.1
3.5
1.3
820
969
982
43 1
1120
363
3.0k0.8
663k248
171+92
2.8+1-2
781k313
142
374
166
147
222
191
207k87
acid amounted to 171 (86-300) mg/day and was similar to that of 207 (142-374) mg/day
recorded for chenodeoxycholic acid.
When patient 4 was re-examined after 6 months the turnover of chenodeoxycholic acid was
found to be almost unchanged. The values encountered for subjects 5 and 6, who had lost 5-6
kg body weight, had decreased by 25 and 35% respectively (Fig. 2). The half-lives of chenodeoxycholic acid were similar in both studies (Table 3), whereas the pool size of the acid had
increased slightly in one subject and decreased in the other two subjects (Fig. 3). In general,
the values of the pool size and the turnover of cholic acid exceeded those obtained for chenodeoxycholic acid. The half-life of cholic acid averaged 1.6 (0.9-2.4) days (Table 3).
The conversion of [14C]deoxycholic acid into trihydroxycholanic acid was studied by
measuring the 14C radioactivity in the latter fraction after elution from the Hostalene column.
u
t
0
1
I
0.4
I
0.8
I
I
I .4
Ratio of pool sizes D / C D
I .2
I
I .6
I
2.0
I
2.4
FIG.1. Ratio of the pool sizes of deoxycholic acid (D) to chenodeoxycholic acid (CD) compared
with the ratio of their concentrations in specimens of duodenal bile of subjects 1-6 (mean and
range).
Turnover of bile acids
187
Although the specific radioactivity of cholic acid tended to increase with time it was in general
less than 1% of that of deoxycholic acid (Table 4). In a similar way the trihydroxycholanic
acid titration peak was analysed for 3H radioactivity in patients who received 3H-labelled
chenodeoxycholic acid. Only trace amounts of 3H were present in the trihydroxycholanic acid
fraction (Table 5).
0
5
4
6
L
Patient no.
FIG.2. Comparison of the turnover values for deoxycholic acid (solid columns) and chenodeoxy
cholic acid (hatched columns) during the first study and the corresponding values recorded for
chenodeoxycholicacid (cross-hatched columns) and cholic acid (open columns) during the second
investigation.
TABLE
3. Half-lives simultaneously recorded for chenodeoxycholic acid and
cholic acid (second study)
Patient
no.
Half-life for chenodeoxycholic acid
(days)
Half-life for cholic acid
(days)
4
5
26
3.2
1.9
2.4
0.9
6
1.6
DISCUSSION
The patients studied in this investigation had a pool size and turnover of chenodeoxycholic
acid which averaged 78 1 mg and 207 mglday respectively. These results are in good agreement
with those of 810 mg and 162 mg/day reported by Vlahcevic, Miller, Fabrar & Swell (1971) for
healthy subjects fed with a natural diet. Under similar experimental conditions, the average
pool size of cholic acid has been reported to be about 1100 mg and the turnover about 300
Kurt Einarsson and Kjell Hellstrom
188
Patient no.
FIG.3. Comparison of the pool size values for deoxycholic acid (solid columns) and chenodeoxycholic acid (hatched columns) during the first study and the corresponding values for chenodeoxycholic acid (cross-hatched columns) and cholic acid (open columns) during the second investigation.
TABLE
4. Percentage of 14C radioactivity recovered in the trihydroxycholanic
acid fraction of duodenal bile after the administration of [14C]deoxycholicacid
Period after the administration of isotope (days)
Patient
no.
1-2
3-4
5-7
8-10
0.22
0.10
0.32
0.22
0.29
1.14
0.47
0.33
0.23
0.10
0.49
0.72
0.36
0.05
0.51
1 .oo
0.22 f0.09
0.56 f:0.40
0.39 f0.28
0.48 f:0.40
1
2
3
4
Mean f:SD
TABLE
5. Percentage of 3H radioactivity recovered in the
trihydroxycholanic acid fraction of duodenal bile after the
administration of [3H]chenodeoxycholicacid
Patient
no.
3
5
6
Mean
Period after administration of isotope (days)
1-2
3-4
5-7
8-10
0
441
2.21
2.96
0.13
2.44
0.39
0
1.97
0.12
2.04
3.15
1.84
0-79
1.77
Turnover of bile acids
189
mg/day (Lindstedt, 1957; Danielsson et al., 1963; Hellstrom & Lindstedt, 1966; Vlahcevic et
al., 1971). On the basis of these reports, the mean value recorded for the formation of deoxycholic acid in the present study (171 +92 mg/day) suggested that half of the cholic acid synthesized was degraded by other pathways. Therefore, in order to arrive at a more exact figure
for this relationship, three of the subjects were re-examined after the administration of radioactive cholic acid and chenodeoxycholic acid. A comparison of the turnover results with those
previously obtained indicated that 36,61 and 25% of the cholic acid was converted into deoxycholic acid in patients 4, 5 and 6. These values can, however, only be regarded as approximate
as two of the patients showed changes in their synthesis rates of chenodeoxycholateduring the
6 months interval and there is the possibility that similar changes in the kinetics of cholic acid
might have occurred. Using a similar technique in their study of five healthy subjects, Hepner,
Hofmann & Thomas (1972) found that the percentage of glycocholic acid converted into
glycodeoxycholic acid was between 5 and 44%.
After repeated administrations of radioactive glycine-conjugated bile acids Hepner et al.
(1972) measured the turnover of all the three bile acids in the bile. Their values for pool size
and turnover of glycodeoxycholic acid averaged 349 mg and 114 mg/day respectively, and were
therefore lower than those (663 mg and 171 mg/day) obtained in the present study for the
deconjugated acids. In addition, their values for pool size and turnover of glycochenodeoxycholic acid exceeded those encountered for glycodeoxycholic acid by more than 50%.
Several authors (Lindstedt, 1957; Hellstrom & Lindstedt, 1966; Vlahcevic et al., 1971;
Hepner et al., 1972) have estimated the pool size of deoxycholic acid indirectly from the values
of the cholic acid pool and the percentage composition of the bile acids in duodenal bile. To
evaluate the correctness of such calculations Hepner et al. (1972) compared the ratios of the
pool sizes of glycocholic acid, glycochenodeoxycholicacid and glycodeoxycholicacid with the
ratios of the concentrations of cholic acid, chenodeoxycholic acid and deoxycholic acid in
specimens of duodenal bile obtained after saponification and analysis by gas-liquid chromatography. The ratios of the concentration of the three bile acids in the bile were found to reflect
closely the ratios of the pool sizes. The fact that such a good correlation was not obtained in the
present study (Fig. 1) demonstrates, however, that the indirect method of measuring the pool
size of deoxycholic acid gives only approximate and occasionally misleading results.
The liver of many species is capable of hydroxylating deoxycholic acid and chenodeoxycholic
acid so that the more polar trihydroxy bile acids are formed. In the rat deoxycholic acid is
quantitatively transformed into cholic acid and chenodeoxycholic acid is to a large extent
hydroxylated in the 6P-position to yield wmuricholic acid (3a,6P,7a-trihydroxycholanicacid)
(Danielsson & Einarsson, 1969). The human liver, however, appears to be less efficient in
performing such transformations. Hellstrom & Sjovall (1961) administered labelled chenodeoxycholic acid orally to a patient with a bile-duct drainage and reported that only 15% of the
label was recovered in material more polar than the parent compound. In a similar study
Hansson &Williams (1971) gave [14C]deoxycholicacid to two patients with a complete bileduct fistula and found that more than 94% of the radioactivity recovered was still attached to
the bile acid they had administered. In a recent investigation (T. Bjorkhem, K. Einarsson &
G. Hellers, unpublished work) the metabolism of taurodeoxycholic acid and of taurochenodeoxycholic acid was studied in human liver homogenates. Under these conditions only 1% of'
taurodeoxycholicacid was hydroxylated to yield taurocholic acid and no significanthydroxylation of taurochenodeoxycholicacid occurred. The present results are in good agreement with
190
Kurt Einarsson and Kjell Hellstrom
these findings, since only trace amounts of radioactivity administered in the form of dihydroxy
bile acids were recovered in the trihydroxycholanic acid fraction of the duodenal bile. Even
allowing for the fact that a substantial part of the radioactivity was probably eliminated in the
faecesthe results indicate that the conversion of dihydroxy into absorbable trihydroxy bile acids
is small in man.
ACKNOWLEDGMENTS
The present investigation was supported by the Swedish Medical Research Council. The assistance of Mrs Lena Rodin, Mrs Kerstin Hedstrom and Miss Margreth Wahlstrom is gratefully
acknowledged. The ethical aspects of this investigation were considered by the Ethics Committee
at the Karolinska Institutet, Stockholm.
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