Yonago Acta medica 1997;40:73–77

An Examination of Alkaline Hydrolyzing Conditions of Conjugated Bile
Acids with Carbonyl Groups
Nobuo Yamaga, Yumiko Nagano and Kazuo Yamada
Department of Biochemistry, Faculty of Medicine, Tottori University, Yonago 683, JapanThe conditions for alkaline hydrolysis of conjugated bile acids with carbonyl groups were
investigated using several oxo bile acids and conjugated bile acids. Under the condition
using a 1 N NaOH solution for 4 h in an autoclave, glycine- and taurine-conjugated bile
acids were hydrolyzed and also, the amounts of oxo bile acids used were recovered without
any significant decomposition.
Key words: alkaline hydrolyzing conditions; capillary gas chromatography; conjugated bile acids;
1 N NaOH solution; oxo bile acids

acids, 7-oxo-3α-hydroxycholanoic acid and 12oxo-3α-hydroxycholanoic acid were grouped
separately (1-a and 1-b in Table 1), since thesebile acids show the same retention time on thepresent capillary column of gas chromatography.
After acidification to pH 1 with 3 N HCl on
ice, bile acids were extracted 3 times with diethyl ether. The extracted bile acids were converted into methyl ester dimethylethylsilyl ether
derivatives as reported previously (Yamaga et al.,1994). The procedure for analysis of urinary
bile acids was described elsewhere (Yamaga etal., 1994). Capillary gas chromatograms andanalytical data of the extracted bile acid derivatives were obtained using a gas chromatograph(Model GC-14A, Shimadzu, Kyoto, Japan)
with a computerized data system (Model C-R4A
Chromatopac, Shimadzu). The gas chromatograph was equipped with a HiCap CBP-1 capillary column (25 m × 0.25 mm I.D., Shimadzu) anda solventless injector. The capillary gas chromatographic conditions were described elsewhere (Yamaga et al., 1994).

Hydrolysis is often applied for the analysis ofbile acids and is performed either in an alkaline
solution or using cholylglycine hydrolase. Alkaline hydrolysis is usually performed with 2 N
NaOH solution for 4 h at 130°C under pressure,but oxo bile acids are vulnerable during this process (Lepage et al., 1978; Yamamoto et al., 1977).
In this study, we examined conditions foralkaline hydrolysis of conjugated bile acids andfound a suitable condition which caused no significant decomposition of oxo bile acids.

Materials and Methods
The unconjugated oxo bile acids and glycineand taurine-conjugated bile acids used in thisstudy were those kept in our laboratory. 7β,12β-Dihydroxycholanoic acid and glyco-7β,12α-dihydroxycholanoic acid as internal standards for analysis by capillary gas chromatography were synthesized as previously described (Arimoto et al., 1982; Yamaga et al., 1994).
These bile acids were estimated to be more than
96% pure by capillary gas chromatography.
Each of four bile acid mixtures, as listed inTable 1, was separately hydrolyzed in a glass
tube by four conditions consisting of a combination of 2.0 mL of 1 N or 2 N NaOH solutions
for a period of 2 h or 4 h in an autoclave (120°C,
1 to 1.2 kgf/cm2 ). Particularly, two oxo bile

Results and Discussion
Oxo bile acids are formed by the action of thedehydrogenase of intestinal flora in mammalian
species (Matern et al., 1975; Eneroth et al.,73

N. Yamaga et al.

1966; Fromm et al., 1980, 1983). Oxo bile
acids such as 12-oxo-3α-hydroxycholanoic
acid and 12-oxo-3β-hydroxycholanoic acidhave been detected as major bile acids in humanfeces (Danielson et al., 1963; Eneroth et al.,
1968; Hill and Aries, 1971). Recently, 12-oxo3α-hydroxycholanoic acid was determined as amajor bile acid among the many species of bile
acids in urine specimens from healthy humans,
and also, no other species of oxo bile acids

except for 12-oxo-3α-hydroxycholanoic acid
were detected in them (Yamaga et al., 1996).
On the other hand, oxo bile acids are reported to
be vulnerable to alkaline hydrolysis (Lepage etal., 1978; Yamamoto et al., 1977), and moreover the chemical mode of oxo bile acids inbiological specimens is unknown. Therefore, itremains a possibility that oxo bile acids in biological specimens have not been measured
completely after alkaline hydrolysis. In thisstudy, suitable conditions for alkaline hydrolysis of conjugated bile acids including conjugated oxo bile acids were investigated using several oxo bile acids and common conjugated bile
acids.
Table 2 summarizes the effects of alkalinehydrolysis on several oxo bile acids, under thefour conditions described in Materials andMethods. Obviously, the oxo bile acids werelabile to alkaline hydrolysis, particularly underconditions which using a 2 N NaOH solution
for 4 h in an autoclave. The four 3-oxo bile
acids (3-oxocholanoic acid, 3-oxo-7α-hydroxycholanoic acid, 3-oxo-12α-hydroxycholanoic
acid and 3-oxo-7α,12α-dihydroxycholanoicacid) were significantly sensitive. Artificial
products from the oxo bile acids were not detected at their peaks on the gas chromatogram,
although Lepage and colleagues (1978) detected some identified and unidentified peaks ofartificial products from corresponding oxo bile
acids. Decreased recoveries of oxo bile acids
were dependent on the concentration of NaOH
solution, but not on the period of hydrolysis.
The recovery for all of oxo bile acid species
including the four labile oxo bile acids mentioned above, was over 80% when a 1 N NaOH
solution was used. The most excellent recovery
for all of oxo bile acid species examined was
obtained under conditions hydrolyzing in a 1 N
NaOH solution for 2 h. These results indicatethat the decomposition of oxo bile acids is
related to the position of carbonyl groups in thesteroid nucleus and to the condition of alkalinehydrolysis.
However, the question remains as to whether glycine- and taurine-conjugated bile acids,
including conjugated oxo bile acids, are completely hydrolyzed by the conditions mentioned

Table 1. The composition and weight of bile
acids used for alkaline hydrolysis1-a) Unconjugated oxo bile acid mixture (µg/tube)
7β,12β-Dihydroxycholanoic acidas internal standard
5.1
3-Oxo-cholanoic acid
5.1
3-Oxo-12α-hydroxycholanoic acid
5.3
3-Oxo-7α-hydroxycholanoic acid4.9
12-Oxo-3α-hydroxycholanoic acid
4.9
3-Oxo-7α,12α-dihydroxycholanoic acid
5.0
7-Oxo-3α,12α-dihydroxycholanoic acid5.2
12-Oxo-3α,7α-dihydroxycholanoic acid
5.11-b) Unconjugated oxo bile acid mixture (µg/tube)
7β,12β-Dihydroxycholanoic acidas internal standard
5.1
3-Oxo-cholanoic acid
5.1
3-Oxo-12α-hydroxycholanoic acid
5.3
3-Oxo-7α-hydroxycholanoic acid
4.9
7-Oxo-3α-hydroxycholanoic acid
5.0
3-Oxo-7α,12α-dihydroxycholanoic acid
5.0
7-Oxo-3α,12α-dihydroxycholanoic acid5.2
12-Oxo-3α,7α-dihydroxycholanoic acid
5.1
2) Glycine-conjugated bile acid mixture [µg (asfree)/tube]
7β,12α-Dihydroxycholanoic acidas internal standard
4.8
Lithocholic acid
3.4
Deoxycholic acid
4.4
Chenodeoxycholic acid
3.5
Cholic acid6.0
[ µg (asfree)/tube]
Glyco-7β,12α-Dihydroxycholanoic acidas internal standard
4.5
Lithocholic acid
2.6
Deoxycholic acid
3.4
Chenodeoxycholic acid
3.5
Cholic acid
4.63) Taurine-conjugated bile acid mixture

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Hydrolysis of conjugated oxo bile acids

Table 2. Effects of alkaline hydrolysis on several unconjugated oxo bile acids
Bile acids

2 h*
1 N NaOH†
2 N NaOHµg (%)µg (%)

1 N NaOHµg (%)

4h
2 N NaOHµg (%)

3-Oxocholanoic acid
5.0 ( 98.0)
3.8 (74.5)
4.8 (94.1)
3.3 (64.7)
3-Oxo-12α-hydroxycholanoic acid
5.2 ( 98.1)
3.8 (71.7)
5.1 (96.2)
3.7 (69.8)
3-Oxo-7α-hydroxycholanoic acid
4.6 ( 93.9)
2.6 (53.1)
4.0 (81.6)
2.4 (53.1)
7-Oxo-3α-hydroxycholanoic acid
4.5 ( 91.8)
4.8 (98.0)
4.7 (95.9)
4.8 (98.0)
12-Oxo-3α-hydroxycholanoic acid
4.9 ( 98.0)
4.7 (94.0)
4.8 (96.0)
4.7 (94.0)
3-Oxo-7α,12α-dihydroxycholanoic acid
5.3 (106.0)
2.7 (54.0)
4.4 (88.0)
2.7 (54.0)
7-Oxo-3α,12α-dihydroxycholanoic acid
4.7 ( 90.4)
4.1 (78.8)
4.2 (80.8)
4.0 (76.9)
12-Oxo-3α,7α-dihydroxycholanoic acid
4.6 ( 90.2)
4.3 (84.3)
4.2 (82.4)
4.2 (82.4)
Data represent mean value of five experiments and numbers in ( ) represent recovery % from the fiveexperiments.
* Period of alkaline hydrolysis.
† Concentration of NaOH solution.

above. For the recovery tests, we used common
glycine- and taurine-conjugated bile acids
instead of conjugated oxo bile acids (unavailable) and their alkaline hydrolysis was performed under 1 N and 2 N NaOH solutions for 2 and
4 h. The results are shown in Table 3. The
glycine-conjugated bile acids were hydrolyzed
sufficiently under all the conditions describedabove. The recoveries of taurine-conjugatedbile acids were almost constant, although theamount recovered was slightly decreased, under

the three conditions except for the 1 N NaOH
solution for 2 h. The decreased recovery is dueto the difficulty of the hydrolysis of taurineconjugated bile acids compared with that ofglycine-conjugated bile acids, and also to theuse of glyco-7β,12α-dihydroxycholanoic acidas an internal standard for analysis of taurineconjugated bile acids. These results indicatethat authentic conjugated bile acids were
obviously hydrolyzed by a 1 N NaOH solution
for 4 h in an autoclave.

Table 3. Effects of alkaline hydrolysis on glycine-conjugated bile acids and taurine-conjugatedbile acids
Conjugatedbile acids
G-LA
G-DOCA
G-CDCA
G-CA
Total

4 h*

2h

1 N†
µg (%)

2N
µg
(%)

1N
µg (%)

3.9 (114.7)
3.7 ( 84.1)
3.3 ( 94.3)
6.0 (100.0)
16.9

3.9 (114.7)
3.8 (102.7)
3.4 ( 97.1)
6.1 (101.7)
17.2

4.1 (120.6)
3.8 ( 86.4)
3.3 ( 94.3)
6.1 (101.7)
17.3

µg

2N
(%)

3.9 (114.7)
4.0 ( 90.9)
3.6 (102.9)
5.9 ( 98.3)
17.4

T-LA
2.2 ( 84.6)
2.4 ( 92.3)
2.2 ( 84.6)
2.2 ( 84.6)
T-DOCA
2.7 ( 79.4)
3.1 ( 91.2)
2.5 ( 73.5)
2.5 ( 73.5)
T-CDCA
2.8 ( 80.0)
2.8 ( 80.0)
2.7 ( 77.1)
3.0 ( 85.7)
T-CA
4.0 ( 87.0)
4.0 ( 87.0)
3.7 ( 80.4)
3.9 ( 84.8)
Total
11.7
12.3
11.1
11.8
Data and numbers in ( ) represent mean value and recovery % from the five experiments, respectively.
* Period of alkaline hydrolysis.
† Concentration of NaOH solution.
CA, 3α,7α,12α-trihydroxycholanoic acid; CDCA, 3α,7α-dihydroxycholanoic acid; DOCA, 3α,12αdihydroxycholanoic acid; G, glycine-conjugated; LA, 3α-hydroxycholanoic acid; T, taurine-conjugated.

75

N. Yamaga et al.

Table 4. The amounts (mg) and compositions (%) of bile acids in 5 mL of urine specimensfrom two healthy subjects (A and B)
Bile acids

Subject A
1 N, 4 h*
2 N, 4 hµg (%)µg (%)

Subject B
1 N, 4 h
2 N, 4 hµg (%)µg(%)

Lithocholic acid
0.98 ( 2.6)
1.17 ( 3.1)
2.68 ( 5.5)
2.73 ( 5.6)
3β-Hydroxychol-5-enoic acid
ND
ND
0.91 ( 1.9)
0.82 ( 1.7)
3α,12β-Dihydroxycholanoic acid
4.93 (13.2)
4.62 (12.3)
0.61 ( 1.3)
0.77 ( 1.6)
3β,7α-Dihydroxycholanoic acid
ND
ND
0.48 ( 1.0)
0.53 ( 1.1)
Deoxycholic acid1.86 ( 5.0)
1.90 ( 5.1)
1.06 ( 2.2)
1.08 ( 2.2)
Chenodeoxycholic acid
ND
ND
2.23 ( 4.6)
2.06 ( 4.2)
12-Oxo-3α-hydroxycholanoic acid
25.37 (67.9) 25.38 (67.7)
3.10 ( 6.4)
3.09 ( 6.4)
Norcholic acid
ND
ND
1.38 ( 2.8)
1.34 ( 2.8)
Ursodeoxycholic acid
0.65 ( 1.7)
0.95 ( 2.3)
5.67 (11.6)
5.81 (11.9)
3β,7β-Dihydroxycholanoic acid
ND
ND
5.25 (10.8)
4.95 (10.1)
3α,7α,12β-Trihydroxycholanoic acid
ND
ND
2.66 ( 5.4)
2.29 ( 4.7)
Cholic acid
2.27 ( 6.1)
2.10 ( 5.6)
3.12 ( 6.4)
2.98 ( 6.1)
3α,7β,12α-Trihydroxycholanoic acid1.31 ( 3.5)
1.39 ( 3.7) 14.98 (30.7)
15.29 (31.4)
7-Oxo-3α,12α-dihydroxycholanoic acid ND
ND
2.83 ( 5.8)
2.60 ( 5.3)
12-Oxo-3α,7α-dihydroxycholanoic acid ND
ND
1.82 ( 3.7)
2.31 ( 4.7)
Total
37.37
37.51
48.78
48.65
Numbers in ( ) represent composition (%) of each bile acid in urine specimens.
* The condition for alkaline hydrolysis. N and h mean the concentration of NaOH solution and a period ofalkaline hydrolysis, respectively.
ND, not detectable.

h in a 1 N NaOH solution instead of a 2 N
NaOH solution, without any significant decomposition of oxo bile acids.

After the extraction of bile acids from urine
specimens (5 mL) of two healthy humans, theextracted bile acids were hydrolyzed separately
by autoclaving for 4 h in 2.0 mL of 1 N and 2 N
NaOH solutions, and then measured. The
amounts and compositions of the urinary bile
acids coincided well in both conditions, and theoxo bile acid identified tentatively by the retention time on the present capillary column of gas
chromatography was only 12-oxo-3α-hydroxycholanoic acid in subject A, and 12-oxo-3αhydroxycholanoic acid, 7-oxo-3α,12α-dihydroxycholanoic acid and 12-oxo-3α,7α-dihydroxycholanoic acid in subject B, as shown in
Table 4. No 3-oxo bile acid was detected ineither specimen. The present data on urine
specimens suggest that glycine- and taurineconjugated bile acids were hydrolyzed undercondition using a 1 N NaOH solution for 4 h in
an autoclave which will not decompose 3-oxobile acids.
We conclude that the conjugated bile acids
and oxo bile acids extracted from biological
specimens are hydrolyzed by autoclaving for 4

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(Received November 25, Accepted December 26, 1996)

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