249
Biochimica et Biophysics Acta, 409 (1975) 249-257 @ Elsevier Scientific Publishing Company, Amsterdam
BBA
-
Printed
in The Netherlands
56676
BILE ACIDS XLVII. 12~HYDROXYLATION OF PRECURSORS BY RABBIT LIVER MICROSOMES
S.S. ALI*
and WILLIAM
Department (U.S.A.) (Received
BILE ACIDS
H. ELLIOTT**
of Biochemistry,
May lst,
OF ALL0
St. Louis University School of Medicine, St. Louis, MO. 63104
1975)
Summary Rabbit liver microsomal preparations fortified with 0.1 mM NADPH effectively promote hydroxylation of [3p- 3H] - or [ 24- ’ 4 C] allochenodeoxycholic acid or [ 501,& - 3Hz ] 5a!-cholestane-& ,7a diol to their respective 12a -hydroxyl derivatives in yields of about 25 or 65% in 60 min. Minor amounts of other products are formed from the diol. The requirements for activity of rabbit liver microsomal l%-hydroxylase resemble those of rat liver microsomes. Of a number of enzyme inhibitors studied only p-chloromercuribenzoate demonstrated a marked ability to inhibit the reaction with either tritiated substrate. There was no difference in the quantity of product produced from the tritiated acid or the ’ 4C-labeled acid. No clear sex difference was found in activity of the enzyme, nor was an appreciable difference noted in activity of the enzyme between mature and immature animals.
Introduction Earlier reports from this laboratory demonstrated the conversion of allochenodeoxycholic acid to allocholic acid by the rat with a cannulated bile duct [l] , or by a rat liver microsomal preparation fortified with NADPH [ 21. From a study of the requirements of the fortified microsomal system for la-hy-
SYstcmatic names of the compounds referred to in the text by their trivial names are as follows: allochenodeoxycholic acid. 3ar.7c4ihydroxy-5osholanic acid; allocholic acid, 3a,7a,l%Mrih~droxy-5olilholanic
acid: all allocholanic
acids are 5asholanic
* On leave from the Department of Biochemistry, ** To whom correspondence should be addressed.
University
acids. of Karachi.
Karachi,
Pakistan.
250
droxylation of allochenodeoxycholate and several substituted 5a: -cholestanes [3,4], it was suggested that the enzyme system may be identical to that concerned with lZff-hydroxylation of 7~-hydroxycholest-4-en-3-one, a precursor of cholic acid. Since a comparison of the activity of this enzyme from rat, rabbit, chicken, and human liver microsomes [5] showed a superior ability of rabbit microsomes to form allocholate from [ 3/3-“H] allochenodeoxycholate, an extended study of the characteristics of the enzyme system of this species was desirable. This paper describes the effects of a number of parameters on the rate of 12~-hydroxylation of [ 3Pe3H J allochenodeoxy~holic acid and [ 5c~,6a! 3Hz ] 5a-cholestane-3cu ,7crdiol by rabbit liver microsomal preparations fortified with NADPH. A preliminary report has appeared [ 61. Methods and Materials Bile acids were separated by acetic acid partition chromatography [7] . The fractions have been designated according to the percentage of benzene in hexane, e.g. Fraction 20-2 represents the second fraction of the eluent containing 20% benzene in hexane. Thin-layer chromatography was carried out [l] with chloroform/methanol/acetic acid (80 : 12 : 3, v/v) [8] or acetone/benzene (1 : 1, v/v); gas-liquid chromato~aphy, mass spectrometry, and radioassay were carried out as described ]9,10,3]. Allocholani~ acids~were prepared from the requisite methyl 5/J-choianates [ll] . The preparation of 5a-cholestane3a ,?a ,12e -trial, 3cu,7a ,120 -trihydroxy-&-cholestanoic acid, and 5a -choles&me-3cu,7a ,l% ,26-tetrol from anhydrocyprinol has been described [ 3,121. The purity of authentic samples was checked by thin-layer and gas-liquid chromatography, melting point, and mass spectrometry, The specific activities of the substrates were: [3fi-3H] allochenodeoxycholic acid, 4.46 * lOa dpm/mg; [24-l 4 C] allochenodeoxycholic acid, 1.39 . lo7 dpm/mg; [5a ,60(-’ H2 ] 501-cholestane-3a ,7ediol, 2.80 - lo* dpm/mg. NADPH was obtained from Sigma Chemical Co., St. Louis; SKF-525A was a product of Smith, Kline, and French, Philadelphia, Pa. Diazald (Aldrich) was used for methylation. ~r~c~iQ~a~io~ of liver ~~~oge~a~e. Rabbits weighing 3--. 4.5 kg were sacrificed by infusion of air in the ear vein. A piece of the right lobe of the liver was removed immediately and homogenized [ 31 at 4°C in freshly prepared Bucher’s medium [3,5], pH 7.4. Microsomal fractions were prepared according to Einarsson [13]. Protein de~rminations were made by the biuret reagent using bovine serum albumin as a standard. In most of the expe~ments, the final protein concentration was adjusted to 6 mg/ml unless otherwise mentioned in the text. Incubation procedure. Two substrates, i.e. [3P-3H] allochenodeoxycholic acid and [ 5a,6&-“Hz ] 5a-cholestane-3a,7a-diol were used in a majority of the incubations; results from incubations with [ 24- 1“C] allochenodeoxycholic acid were compared with those from the tritiated acid. Each series of incubations was carried out in duplicate in air with shaking at 37°C with 3 ml of microsomal preparation, 3 pmol of NADPH in 0.1 ml of homogenizing medium, and 165 or 175 nmol of substrate in 50 ~1 of methanol for periods from 10 to 60 min. Microsomal preparations heated at 100°C for 15 min before incubation with the substrate and added NADPH served as controls. The reactions were
251
quenched with 10 volumes of 95% ethanol; the precipitates were filtered, washed with ethanol, and the filtrate was evaporated in a slow stream of Nz . The contents were redissolved in 10 ml of methanol for further analysis. Analysis of incubation mixture. An aliquot (0.1 ml) of the incubation mixture was analyzed by thin-layer chromatography on plates (20 X 20 cm) coated with silica gel H (0.25 mm thickness). Authentic standards were applied along with the incubation mixture in each analysis. After development of the plate, appropriate regions corresponding to substrate and the hydroxylated products were scraped from the plate and assayed for radioactivity. Results Identification of the 12a-hydroxylated products (a) Identification of allocholic ucid. After incubation of [ 3fl- 3H] allochenodeoxycholic acid (175 nmol) for 60 min with hepatic microsomes from male rabbits, 25.4% of the radioactivity appeared on the thin-layer chromatogram in the region associated with allocholic acid. Following incubation of comparable quantities of [ 3fl-3H] - or [24- ’ 4C] allochenodeoxycholic acid for 30 min, equivalent amounts of radioactivity appeared in the region of the product (10.50 and 10.42%, respectively), indicating that no appreciable loss of 3H had occurred from the 3/3-position in these experiments. No significant amount of radioactivity was associated with allocholic acid after 60 min incubation of either labeled acid with heated microsomes. Mixtures from two 60-mm incubations were pooled and the components separated by acetic acid partition chromatography. The substrate was recovered in Fractions 20-2 to 40-3, mixed with 50 mg of authentic compound, methylated with freshly prepared diazomethane and identified by coincident elution of mass and radioactivity in Fractions O-l to 10-l [14]. The residues from Fractions SO-1 to 80-3 were combined, mixed with 50 mg of authentic allocholic acid, and methylated with diazomethane. After rechromatography methyl allocholate (Fractions 40-1, 60-l and 60-2,49.1 mg) gave the following specific activities after crystallization from methanol/acetone (1 : 1, v/v), methanol/acetone (1 : 20, v/v) and methanol, respectively: 1.14 - 10’) 1.11 * 10’ and 1.13 * 10’ dpm/mg. (b) Identification of Sa-cholestane-3cq7aJ2cu-triol. After incubation of [ & ,601-3Hz ] 5e-cholestane-3a ,7cr-diol (165 nmol) with microsomes for 60 min about 70% of the tritium appeared on the chromatogram in the area with 5ar-cholestane-3cw ,7a ,l% -trial. No significant activity appeared in this area from the heated controls. Samples from the incubations were pooled, fractionated by acetic acid partition chromatography, and the material in Fractions O-2 to O-4 was combined, and the solvent evaporated under Nz . An aliquot (l/10) was removed, converted to the trimethylsilyl ether [9], and the product analyzed by gas chromatography on columns of 3% QF-1, 3% OV-17, and 3% OV-210. Retention times of 0.44, 0.46 and 0.42, relative to the bis-trimethylsilyl ether of methyl deoxycholate were identical with those obtained from this derivative of authentic triol. By gas chromatography-mass spectrometry (1% SE-30) comparable intensities for the following fragment ions were observed for the trimethylsilyl ether of the authentic trio1 and of the radioactive metabolite: m/e
252
636, M; m/e 546 (M-90); 531 [M-(90 + 15)] ; 456 [M-(2 X 90)] ; (base peak); 366 [M-(3 X go)]; 351 [M-(3 X 90 + 15)] ; 343 [M-(2 X 90 + side chain)]; 253 [M-(3 X 90 + side chain)] ; 259; and 211, typical of a trisubstituted sterol nucleus [lo] . Effect of time, substrate concentration, and NADPH on 12a-hydroxylation The superiority of 501-cholestane-3a ,7a-diol to allochenodeoxycholate for these enzymatic studies is shown in Figs l-3. Over a period of 60 min the yield of l%-hydroxylated product was 3-3.5-fold greater from the diol than the acid (Fig. 1). A linear response up to 20 min was found for the diol after which the rate of trio1 formation plateaued. The rate of 12e-hydroxylation of the tritiated acid was linear ,from 10 to 60 min; no measurements were made prior to 10 min. With regard to substrate concentration the maximum reaction occurred at approx. 300 nmol for either substrate (Fig. 2). The maximal effect of protein concentration for either substrate was 15-20 mg/ml of protein (Fig. 3). However, since significant amounts of 12e_-hydroxylation take place at lower protein concentration i.e. 5-10 mg/ml protein, other studies were performed using 5-6 mg/ml protein for such sequence of reaction. The requirement for NADPH was satisfied at 0.1 mM for either substrate (Fig. 4). Experiments similar to those of Fig. 1 were carried out with microsomal preparations of rabbit heart or kidney, but no 12e-hydroxylated product was detected, suggesting the absence of this enzyme system in these tissues. Effect of freezing and thawing of microsomal preparations The liver from a male rabbit was excised, retained at -20°C and at various intervals a portion of liver was removed, thawed, and assayed for 12a-hydroxylase activity with tritiated samples of the acid and the diol. The enzymatic activity for 13 and 30 days was reduced to 50% for allochenodeoxycho-
d
- 100
too-
I 80Z 00g
40-
g
20-
-
IO
20
TIME
60
30 (min)
I
I
MI0
300 Substrate(nmol
500
20
3 4
703
I
Fig. 1. Relation of yield of product to period of incubation. The substrates 3/S3H allochenodeoxycholic acid (e), 175 nmol. and 50,6w3H2 Wx-cholestane-3o.7cu-diol (o), 165 nmol, were incubated with 3 ml of microsomal fraction (18 mg protein) and 3 pmol NADPH for varying periods of time. Fig. 2. Relation of yield of product to amount of substrate. Varying amounts of substrate (3p-3H allochenodeoxycholic acid (ACDC) or 5cz.&~-~H~ 5osholestane-3or,7a-diol (Diol)) were incubated for 30 min with 3 ml of microsomal fraction and 3 I.cmol of NADPH.
100
1 60
1
I
5
1
1
1
10 15 20 Protein img/mlI
I
25 NAIIPH ImM)
Fig. 3. Relation of yield of product to quantity of microsomal protein. Varying amounts of microsomal fraction were incubated for 30 min with 175 nmol of 3p-3H allochenodeoxycholate (ACDC) or 165 nmol of 50,60-3H2 5wcholestane-3a,7a_diol (Dial) with 3 pmol of NADPH. Fig. 4. Relation of yield of product to quantity of added NADPH. Varying amounts of NADPH were added to 175 nmol of txitiated bile acid (ACDC) or 166 nmol of tritiated diol (Dial) with 3 ml of microsomal tiaction and incubated for 30 min.
late and 63-73s for the diol; after 57 days the activities were 30 and 42%, respectively, for the substrates compared to the original fresh liver. In another experiment in which the liver remained frozen for 216 days, the activities for the substrates were reduced to 16 and 58%, respectively. In all other experiments freshly prepared microsomal fractions were utilized. Effect of inhibitors and metal complexing agents Results with microsomal preparations from two male rabbits (Table I) show that metal chelating agents such as o-phenanthroline and EDTA did not inhibit 12~ -hydroxylation to a significant extent. Striking inhibition (92-94s) was only noticed with 1 mM p-chloromercuribenzoate; KCN affected the reaction only to the extent of ll-14%. Addition of 10 mM of glutathione to the incubation mixture enhanced the action up to 75% for allochenodeoxycholic acid and 16% for the diol. When 1 mM of p-chloromercuribenzoate was added to 10 mM glutathione, no inhibition was noted with either of the substrates. SKF 525A was not inhibitory at 0.1 mM but was active at 1 mM to the extent of 45 and 12% for allochenodeoxycholic acid and the diol, respectively. Addition of 10 mM glutathione to 1 mM SKF 525A produced 23 and 10% inhibition for allochenodeoxycholate and the diol, respectively. In a preliminary experiment CO was passed for 5, 10 or 20 s through one-half of the volume of the microsomal fraction used in the previous experiments and incubated with [3fl-3H] allochenodeoxycholic acid for 20 min. The percentages of allocholic acid formed were not significantly different from that of the untreated control. Hence, no significant inhibition was noted in the process of 12ar-hydroxylation. Influence of sex and age Hepatic microsomal
preparations
from
4 mature
rabbits
(2 male and 2
254
TABLE
I
EFFECT
OF
INHIBITORS
concentra-
Addition
Allocholic
Acid
5a-Cholestane-Sol,
tion
70,
(mM)
__
None*
nm01
Inhibition
formed
(%)
40.78
12a-trio1
nmol
Inhibition
formed
(“6)
0
105.9
Ethanol
0.6
31.5
23
102.5
0 3
KCN
0.1
39.2
4
105.4
0
1.0
35.2
14
0.1
39.6
3
106.6
94.5
11 -1
1.0
36.9
9
103.3
NaN3
1.0
37.8
7
98.5
p-Chloromercuribenzoatr
1.0
o-Phenanthroline
0.5
EDTA
1.0
None
2.63
* *
94
6.6
35.2
14
100.98
50.8
--2 4
Glutathione
6.28 19.97
5 -7 0
70.1
-4 45 -75
0.89
1
70.3
0
61.4
12
81.2
92
-16
5.68
92
10
+
1
p-chloromercuribenzoate Glutathione
11.88
1.0 10
p-Chloromercuribenzoate Glutathionr
0.1
7 94
113.3
0
11.41
SKF-525A
2
+
23.73
-108
5
73.4
10
SKF-525A
1
8.73
23
62.9
10
co OS
11.34
5s
13.54
-19
_
12.64
-11
_
10s 20 *
Incubation
cholate **
-
s
(175
Incubation
of male
rabbit,
time. nmol)
30
min;
protein
12.11 concentration,
50i_cholestane-301,7o-diol
time.
20
3.75
kg.
min;
protein
(165
concentration.
5 mg/ml; nmol); 4.8
-7 substrate
weight
of male
mg/mI;
substrate
concentrations. rabbit.
4.75
concentration
allochenodeoxykg. as above:
weight
female animals, 3--4 kg) and 4 immature rabbits (2 male and 2 female animals of approx. 8 weeks of age, 1.6-2.0 kg) were incubated with [3fl-3H] allochenodeoxycholic acid and with [ 5a ,601-‘Hz ] 5a -cholestane-3a ,7a-diol (Table II). A large variation in 12c~-hydroxylase activity occurred between male and females as well as in the animals of two age groups, although the diol consistently provided more product. Discussion Since cholic and deoxycholic acids are the major bile acids in the rabbit an active l&-steroid hydroxylase must be presumed. Chenodeoxyv51, cholate has been detected in low concentration in fistula bile [16] , feces [ 171,
255 TABLE II INFLUENCE
OF SEX AND AGE ON lPc+HYDROXYLATION
Standard assay conditions were used. The amount of protein was 4.8 mg/ml for 30 min with 175 nmol of alloehenodeoxycholate or 165 nmol of Sa-cholestane-3cu,7~-diol. Animal
Allocholic Acid formed
~-Cholestane-3~.7a.l2~-trio1
%
nmol
11.8 16.1
20.7 28.2
(A)
(R)
fBf
46.1 56.9
76.1 93.9
Mature female
(A) (JJ)
11.7 21.4
20.5 37.5
(A) (B)
47.0 62.5
77.6 103.1
Immature male
(A) fR)
14.5 20.7
25.4 36.2
(A) (B)
53.8 61.3
88.8 101.1
Immature female
(A) fRt
8.5 18.2
14.9 31.9
(A) tB)
35.0 55.1
57.8 90.9
Mature male
(4
%
formed
nmo1
and bile of the germ-free rabbit [18], but not in normal rabbit bile [19]. Synthesis of chenodeoxycholate was demonstrated by perfusion of [4-I “C] 7ff-hydroxy~holesterol through rabbit liver, but choIate was the major bile acid [ZO] . Chenodeoxy~holate is metabolized by the rabbit to 7-oxolithocholate and ursodeoxycholate [19], but not to cholate [5 1. Alternately allochenodeoxycholate, a metabolite of cholestanol in the rat [14,21], can be converted to allocholate by the rabbit [ 51 (Fig. 1) in quantities 3-6 times greater than produced by the rat in a comparable period. This may explain in part why ~loch~nodeoxycholate has not been found in rabbit. Allocholate is a metabolite of [ 4- I 4 C] 7a -hydroxycholesterol in the rabbit with a bile fistula [22] and is converted by intestinal bacteria to allodeoxycholate, a normal constituent of rabbit bile and gallstones [17,23,24]. The formation of allocholate from cholestanol in the rat [25-,271, gerbil [28], rabbit 122,231, and other species [ll] has been investigated, and 5a-cholestane-3a ,7cr-dial has been suggested as the point of bifurcation in the formation of di- and trihydroxy allo acids [26,27]. Thus, 12a-hydroxylation of this diol should provide 5a-cholestane-3a! ,7c~,l2a -trial, an intermediate in the formation of allocholate. The plateau found (Fig. 1) after 30 min in the plot of product formation vs period of incubation suggests product inhibition or the formation of other products. Investigation of other radioactive fractions in the thin-layer chromatograms after incubations for 60 min showed the presence of companion products whose identities will be the subject of a subsequent manuscript. The requirements of rabbit liver microsomal 12a-steroid hydroxylase generally resemble those of rat liver [3,13] ; i.e. NADPH is a necessary cofactor [3 1 at a level of 0.1 mM (Fig. 3). Of a number of substances tested (Table I), p-chloromercuribenzoate was the only effective inhibitor for the enzyme preparation. The drug SKF-525A showed some activity at 1.0 mM which was mediated by 10 mM glutathione. Significant reduction of enzyme activity with p-chloromercuribenzoate and SKF-525A and its recovery by glutathione suggests the involvement of sulfhydryl group(s) in the 12a-hydroxylase. It is to be
256
noted that the effect of p-chloromercuribenzoate and SKF-525 on 120:-hydroxylation of 5a_cholestane-3cu ,7cu-dial is similar to that reported for 7~ -hydroxychol~st-4-en-3-one [ 131. CO had little effect on the reaction. Bjijrkhem and Danielsson [29] have reviewed the low sensitivity of rat liver 12&-hydroxylase toward CO, and reported an inhibition of about 30% in an atmosphere of 40% CO, 4% Nz and 56% N2. Ichikawa and Mason [30] reported approx. 1.5 nmol of P-450/mg protein for rabbit microsomes. Clearly, the role of cytochrome P-450 in 12a-hydroxylation is still unresolved. The activity of hepatic 12cu-hydroxylase after freezing and thawing is significantly decreased after 2-7 weeks storage at -20°C. Shefer et al. [ 311 reported that rat liver microsomes stored at -15°C for 1 month lost 50% of their activity. Omura and Sato [ 321 used rabbit microsomal suspensions stored at 4” C for 2---3 days. Rabbits of different age or sex (Table II) afforded variable activity of microsomal 12cu-hydroxylase, demonstrating that a single microsomal preparation is essential for each set of experiments. Einarsson f13] reported a 40% increase in 12cu-hydroxylation of 7cu-hydroxycholest-4-en-3-one for the adult male rat over the immature rat, and a 50% higher activity in the adult male rat over the adult female rat. Acknowledgements This investigation was supported by U.S. Public Health Service Grant HE-07878. The authors are pleased to acknowledge the expert technical assistance of John Branca, Eric Stephenson and William Frasure. Dr Burton W. No11 kindly provided a sample of [24-l 4 C J allochenodeoxycholic acid. References 1
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Biochimica et Biophysics Acta, 409 (1975) 249-257 @ Elsevier Scientific Publishing Company, Amsterdam
BBA
-
Printed
in The Netherlands
56676
BILE ACIDS XLVII. 12~HYDROXYLATION OF PRECURSORS BY RABBIT LIVER MICROSOMES
S.S. ALI*
and WILLIAM
Department (U.S.A.) (Received
BILE ACIDS
H. ELLIOTT**
of Biochemistry,
May lst,
OF ALL0
St. Louis University School of Medicine, St. Louis, MO. 63104
1975)
Summary Rabbit liver microsomal preparations fortified with 0.1 mM NADPH effectively promote hydroxylation of [3p- 3H] - or [ 24- ’ 4 C] allochenodeoxycholic acid or [ 501,& - 3Hz ] 5a!-cholestane-& ,7a diol to their respective 12a -hydroxyl derivatives in yields of about 25 or 65% in 60 min. Minor amounts of other products are formed from the diol. The requirements for activity of rabbit liver microsomal l%-hydroxylase resemble those of rat liver microsomes. Of a number of enzyme inhibitors studied only p-chloromercuribenzoate demonstrated a marked ability to inhibit the reaction with either tritiated substrate. There was no difference in the quantity of product produced from the tritiated acid or the ’ 4C-labeled acid. No clear sex difference was found in activity of the enzyme, nor was an appreciable difference noted in activity of the enzyme between mature and immature animals.
Introduction Earlier reports from this laboratory demonstrated the conversion of allochenodeoxycholic acid to allocholic acid by the rat with a cannulated bile duct [l] , or by a rat liver microsomal preparation fortified with NADPH [ 21. From a study of the requirements of the fortified microsomal system for la-hy-
SYstcmatic names of the compounds referred to in the text by their trivial names are as follows: allochenodeoxycholic acid. 3ar.7c4ihydroxy-5osholanic acid; allocholic acid, 3a,7a,l%Mrih~droxy-5olilholanic
acid: all allocholanic
acids are 5asholanic
* On leave from the Department of Biochemistry, ** To whom correspondence should be addressed.
University
acids. of Karachi.
Karachi,
Pakistan.
250
droxylation of allochenodeoxycholate and several substituted 5a: -cholestanes [3,4], it was suggested that the enzyme system may be identical to that concerned with lZff-hydroxylation of 7~-hydroxycholest-4-en-3-one, a precursor of cholic acid. Since a comparison of the activity of this enzyme from rat, rabbit, chicken, and human liver microsomes [5] showed a superior ability of rabbit microsomes to form allocholate from [ 3/3-“H] allochenodeoxycholate, an extended study of the characteristics of the enzyme system of this species was desirable. This paper describes the effects of a number of parameters on the rate of 12~-hydroxylation of [ 3Pe3H J allochenodeoxy~holic acid and [ 5c~,6a! 3Hz ] 5a-cholestane-3cu ,7crdiol by rabbit liver microsomal preparations fortified with NADPH. A preliminary report has appeared [ 61. Methods and Materials Bile acids were separated by acetic acid partition chromatography [7] . The fractions have been designated according to the percentage of benzene in hexane, e.g. Fraction 20-2 represents the second fraction of the eluent containing 20% benzene in hexane. Thin-layer chromatography was carried out [l] with chloroform/methanol/acetic acid (80 : 12 : 3, v/v) [8] or acetone/benzene (1 : 1, v/v); gas-liquid chromato~aphy, mass spectrometry, and radioassay were carried out as described ]9,10,3]. Allocholani~ acids~were prepared from the requisite methyl 5/J-choianates [ll] . The preparation of 5a-cholestane3a ,?a ,12e -trial, 3cu,7a ,120 -trihydroxy-&-cholestanoic acid, and 5a -choles&me-3cu,7a ,l% ,26-tetrol from anhydrocyprinol has been described [ 3,121. The purity of authentic samples was checked by thin-layer and gas-liquid chromatography, melting point, and mass spectrometry, The specific activities of the substrates were: [3fi-3H] allochenodeoxycholic acid, 4.46 * lOa dpm/mg; [24-l 4 C] allochenodeoxycholic acid, 1.39 . lo7 dpm/mg; [5a ,60(-’ H2 ] 501-cholestane-3a ,7ediol, 2.80 - lo* dpm/mg. NADPH was obtained from Sigma Chemical Co., St. Louis; SKF-525A was a product of Smith, Kline, and French, Philadelphia, Pa. Diazald (Aldrich) was used for methylation. ~r~c~iQ~a~io~ of liver ~~~oge~a~e. Rabbits weighing 3--. 4.5 kg were sacrificed by infusion of air in the ear vein. A piece of the right lobe of the liver was removed immediately and homogenized [ 31 at 4°C in freshly prepared Bucher’s medium [3,5], pH 7.4. Microsomal fractions were prepared according to Einarsson [13]. Protein de~rminations were made by the biuret reagent using bovine serum albumin as a standard. In most of the expe~ments, the final protein concentration was adjusted to 6 mg/ml unless otherwise mentioned in the text. Incubation procedure. Two substrates, i.e. [3P-3H] allochenodeoxycholic acid and [ 5a,6&-“Hz ] 5a-cholestane-3a,7a-diol were used in a majority of the incubations; results from incubations with [ 24- 1“C] allochenodeoxycholic acid were compared with those from the tritiated acid. Each series of incubations was carried out in duplicate in air with shaking at 37°C with 3 ml of microsomal preparation, 3 pmol of NADPH in 0.1 ml of homogenizing medium, and 165 or 175 nmol of substrate in 50 ~1 of methanol for periods from 10 to 60 min. Microsomal preparations heated at 100°C for 15 min before incubation with the substrate and added NADPH served as controls. The reactions were
251
quenched with 10 volumes of 95% ethanol; the precipitates were filtered, washed with ethanol, and the filtrate was evaporated in a slow stream of Nz . The contents were redissolved in 10 ml of methanol for further analysis. Analysis of incubation mixture. An aliquot (0.1 ml) of the incubation mixture was analyzed by thin-layer chromatography on plates (20 X 20 cm) coated with silica gel H (0.25 mm thickness). Authentic standards were applied along with the incubation mixture in each analysis. After development of the plate, appropriate regions corresponding to substrate and the hydroxylated products were scraped from the plate and assayed for radioactivity. Results Identification of the 12a-hydroxylated products (a) Identification of allocholic ucid. After incubation of [ 3fl- 3H] allochenodeoxycholic acid (175 nmol) for 60 min with hepatic microsomes from male rabbits, 25.4% of the radioactivity appeared on the thin-layer chromatogram in the region associated with allocholic acid. Following incubation of comparable quantities of [ 3fl-3H] - or [24- ’ 4C] allochenodeoxycholic acid for 30 min, equivalent amounts of radioactivity appeared in the region of the product (10.50 and 10.42%, respectively), indicating that no appreciable loss of 3H had occurred from the 3/3-position in these experiments. No significant amount of radioactivity was associated with allocholic acid after 60 min incubation of either labeled acid with heated microsomes. Mixtures from two 60-mm incubations were pooled and the components separated by acetic acid partition chromatography. The substrate was recovered in Fractions 20-2 to 40-3, mixed with 50 mg of authentic compound, methylated with freshly prepared diazomethane and identified by coincident elution of mass and radioactivity in Fractions O-l to 10-l [14]. The residues from Fractions SO-1 to 80-3 were combined, mixed with 50 mg of authentic allocholic acid, and methylated with diazomethane. After rechromatography methyl allocholate (Fractions 40-1, 60-l and 60-2,49.1 mg) gave the following specific activities after crystallization from methanol/acetone (1 : 1, v/v), methanol/acetone (1 : 20, v/v) and methanol, respectively: 1.14 - 10’) 1.11 * 10’ and 1.13 * 10’ dpm/mg. (b) Identification of Sa-cholestane-3cq7aJ2cu-triol. After incubation of [ & ,601-3Hz ] 5e-cholestane-3a ,7cr-diol (165 nmol) with microsomes for 60 min about 70% of the tritium appeared on the chromatogram in the area with 5ar-cholestane-3cw ,7a ,l% -trial. No significant activity appeared in this area from the heated controls. Samples from the incubations were pooled, fractionated by acetic acid partition chromatography, and the material in Fractions O-2 to O-4 was combined, and the solvent evaporated under Nz . An aliquot (l/10) was removed, converted to the trimethylsilyl ether [9], and the product analyzed by gas chromatography on columns of 3% QF-1, 3% OV-17, and 3% OV-210. Retention times of 0.44, 0.46 and 0.42, relative to the bis-trimethylsilyl ether of methyl deoxycholate were identical with those obtained from this derivative of authentic triol. By gas chromatography-mass spectrometry (1% SE-30) comparable intensities for the following fragment ions were observed for the trimethylsilyl ether of the authentic trio1 and of the radioactive metabolite: m/e
252
636, M; m/e 546 (M-90); 531 [M-(90 + 15)] ; 456 [M-(2 X 90)] ; (base peak); 366 [M-(3 X go)]; 351 [M-(3 X 90 + 15)] ; 343 [M-(2 X 90 + side chain)]; 253 [M-(3 X 90 + side chain)] ; 259; and 211, typical of a trisubstituted sterol nucleus [lo] . Effect of time, substrate concentration, and NADPH on 12a-hydroxylation The superiority of 501-cholestane-3a ,7a-diol to allochenodeoxycholate for these enzymatic studies is shown in Figs l-3. Over a period of 60 min the yield of l%-hydroxylated product was 3-3.5-fold greater from the diol than the acid (Fig. 1). A linear response up to 20 min was found for the diol after which the rate of trio1 formation plateaued. The rate of 12e-hydroxylation of the tritiated acid was linear ,from 10 to 60 min; no measurements were made prior to 10 min. With regard to substrate concentration the maximum reaction occurred at approx. 300 nmol for either substrate (Fig. 2). The maximal effect of protein concentration for either substrate was 15-20 mg/ml of protein (Fig. 3). However, since significant amounts of 12e_-hydroxylation take place at lower protein concentration i.e. 5-10 mg/ml protein, other studies were performed using 5-6 mg/ml protein for such sequence of reaction. The requirement for NADPH was satisfied at 0.1 mM for either substrate (Fig. 4). Experiments similar to those of Fig. 1 were carried out with microsomal preparations of rabbit heart or kidney, but no 12e-hydroxylated product was detected, suggesting the absence of this enzyme system in these tissues. Effect of freezing and thawing of microsomal preparations The liver from a male rabbit was excised, retained at -20°C and at various intervals a portion of liver was removed, thawed, and assayed for 12a-hydroxylase activity with tritiated samples of the acid and the diol. The enzymatic activity for 13 and 30 days was reduced to 50% for allochenodeoxycho-
d
- 100
too-
I 80Z 00g
40-
g
20-
-
IO
20
TIME
60
30 (min)
I
I
MI0
300 Substrate(nmol
500
20
3 4
703
I
Fig. 1. Relation of yield of product to period of incubation. The substrates 3/S3H allochenodeoxycholic acid (e), 175 nmol. and 50,6w3H2 Wx-cholestane-3o.7cu-diol (o), 165 nmol, were incubated with 3 ml of microsomal fraction (18 mg protein) and 3 pmol NADPH for varying periods of time. Fig. 2. Relation of yield of product to amount of substrate. Varying amounts of substrate (3p-3H allochenodeoxycholic acid (ACDC) or 5cz.&~-~H~ 5osholestane-3or,7a-diol (Diol)) were incubated for 30 min with 3 ml of microsomal fraction and 3 I.cmol of NADPH.
100
1 60
1
I
5
1
1
1
10 15 20 Protein img/mlI
I
25 NAIIPH ImM)
Fig. 3. Relation of yield of product to quantity of microsomal protein. Varying amounts of microsomal fraction were incubated for 30 min with 175 nmol of 3p-3H allochenodeoxycholate (ACDC) or 165 nmol of 50,60-3H2 5wcholestane-3a,7a_diol (Dial) with 3 pmol of NADPH. Fig. 4. Relation of yield of product to quantity of added NADPH. Varying amounts of NADPH were added to 175 nmol of txitiated bile acid (ACDC) or 166 nmol of tritiated diol (Dial) with 3 ml of microsomal tiaction and incubated for 30 min.
late and 63-73s for the diol; after 57 days the activities were 30 and 42%, respectively, for the substrates compared to the original fresh liver. In another experiment in which the liver remained frozen for 216 days, the activities for the substrates were reduced to 16 and 58%, respectively. In all other experiments freshly prepared microsomal fractions were utilized. Effect of inhibitors and metal complexing agents Results with microsomal preparations from two male rabbits (Table I) show that metal chelating agents such as o-phenanthroline and EDTA did not inhibit 12~ -hydroxylation to a significant extent. Striking inhibition (92-94s) was only noticed with 1 mM p-chloromercuribenzoate; KCN affected the reaction only to the extent of ll-14%. Addition of 10 mM of glutathione to the incubation mixture enhanced the action up to 75% for allochenodeoxycholic acid and 16% for the diol. When 1 mM of p-chloromercuribenzoate was added to 10 mM glutathione, no inhibition was noted with either of the substrates. SKF 525A was not inhibitory at 0.1 mM but was active at 1 mM to the extent of 45 and 12% for allochenodeoxycholic acid and the diol, respectively. Addition of 10 mM glutathione to 1 mM SKF 525A produced 23 and 10% inhibition for allochenodeoxycholate and the diol, respectively. In a preliminary experiment CO was passed for 5, 10 or 20 s through one-half of the volume of the microsomal fraction used in the previous experiments and incubated with [3fl-3H] allochenodeoxycholic acid for 20 min. The percentages of allocholic acid formed were not significantly different from that of the untreated control. Hence, no significant inhibition was noted in the process of 12ar-hydroxylation. Influence of sex and age Hepatic microsomal
preparations
from
4 mature
rabbits
(2 male and 2
254
TABLE
I
EFFECT
OF
INHIBITORS
concentra-
Addition
Allocholic
Acid
5a-Cholestane-Sol,
tion
70,
(mM)
__
None*
nm01
Inhibition
formed
(%)
40.78
12a-trio1
nmol
Inhibition
formed
(“6)
0
105.9
Ethanol
0.6
31.5
23
102.5
0 3
KCN
0.1
39.2
4
105.4
0
1.0
35.2
14
0.1
39.6
3
106.6
94.5
11 -1
1.0
36.9
9
103.3
NaN3
1.0
37.8
7
98.5
p-Chloromercuribenzoatr
1.0
o-Phenanthroline
0.5
EDTA
1.0
None
2.63
* *
94
6.6
35.2
14
100.98
50.8
--2 4
Glutathione
6.28 19.97
5 -7 0
70.1
-4 45 -75
0.89
1
70.3
0
61.4
12
81.2
92
-16
5.68
92
10
+
1
p-chloromercuribenzoate Glutathione
11.88
1.0 10
p-Chloromercuribenzoate Glutathionr
0.1
7 94
113.3
0
11.41
SKF-525A
2
+
23.73
-108
5
73.4
10
SKF-525A
1
8.73
23
62.9
10
co OS
11.34
5s
13.54
-19
_
12.64
-11
_
10s 20 *
Incubation
cholate **
-
s
(175
Incubation
of male
rabbit,
time. nmol)
30
min;
protein
12.11 concentration,
50i_cholestane-301,7o-diol
time.
20
3.75
kg.
min;
protein
(165
concentration.
5 mg/ml; nmol); 4.8
-7 substrate
weight
of male
mg/mI;
substrate
concentrations. rabbit.
4.75
concentration
allochenodeoxykg. as above:
weight
female animals, 3--4 kg) and 4 immature rabbits (2 male and 2 female animals of approx. 8 weeks of age, 1.6-2.0 kg) were incubated with [3fl-3H] allochenodeoxycholic acid and with [ 5a ,601-‘Hz ] 5a -cholestane-3a ,7a-diol (Table II). A large variation in 12c~-hydroxylase activity occurred between male and females as well as in the animals of two age groups, although the diol consistently provided more product. Discussion Since cholic and deoxycholic acids are the major bile acids in the rabbit an active l&-steroid hydroxylase must be presumed. Chenodeoxyv51, cholate has been detected in low concentration in fistula bile [16] , feces [ 171,
255 TABLE II INFLUENCE
OF SEX AND AGE ON lPc+HYDROXYLATION
Standard assay conditions were used. The amount of protein was 4.8 mg/ml for 30 min with 175 nmol of alloehenodeoxycholate or 165 nmol of Sa-cholestane-3cu,7~-diol. Animal
Allocholic Acid formed
~-Cholestane-3~.7a.l2~-trio1
%
nmol
11.8 16.1
20.7 28.2
(A)
(R)
fBf
46.1 56.9
76.1 93.9
Mature female
(A) (JJ)
11.7 21.4
20.5 37.5
(A) (B)
47.0 62.5
77.6 103.1
Immature male
(A) fR)
14.5 20.7
25.4 36.2
(A) (B)
53.8 61.3
88.8 101.1
Immature female
(A) fRt
8.5 18.2
14.9 31.9
(A) tB)
35.0 55.1
57.8 90.9
Mature male
(4
%
formed
nmo1
and bile of the germ-free rabbit [18], but not in normal rabbit bile [19]. Synthesis of chenodeoxycholate was demonstrated by perfusion of [4-I “C] 7ff-hydroxy~holesterol through rabbit liver, but choIate was the major bile acid [ZO] . Chenodeoxy~holate is metabolized by the rabbit to 7-oxolithocholate and ursodeoxycholate [19], but not to cholate [5 1. Alternately allochenodeoxycholate, a metabolite of cholestanol in the rat [14,21], can be converted to allocholate by the rabbit [ 51 (Fig. 1) in quantities 3-6 times greater than produced by the rat in a comparable period. This may explain in part why ~loch~nodeoxycholate has not been found in rabbit. Allocholate is a metabolite of [ 4- I 4 C] 7a -hydroxycholesterol in the rabbit with a bile fistula [22] and is converted by intestinal bacteria to allodeoxycholate, a normal constituent of rabbit bile and gallstones [17,23,24]. The formation of allocholate from cholestanol in the rat [25-,271, gerbil [28], rabbit 122,231, and other species [ll] has been investigated, and 5a-cholestane-3a ,7cr-dial has been suggested as the point of bifurcation in the formation of di- and trihydroxy allo acids [26,27]. Thus, 12a-hydroxylation of this diol should provide 5a-cholestane-3a! ,7c~,l2a -trial, an intermediate in the formation of allocholate. The plateau found (Fig. 1) after 30 min in the plot of product formation vs period of incubation suggests product inhibition or the formation of other products. Investigation of other radioactive fractions in the thin-layer chromatograms after incubations for 60 min showed the presence of companion products whose identities will be the subject of a subsequent manuscript. The requirements of rabbit liver microsomal 12a-steroid hydroxylase generally resemble those of rat liver [3,13] ; i.e. NADPH is a necessary cofactor [3 1 at a level of 0.1 mM (Fig. 3). Of a number of substances tested (Table I), p-chloromercuribenzoate was the only effective inhibitor for the enzyme preparation. The drug SKF-525A showed some activity at 1.0 mM which was mediated by 10 mM glutathione. Significant reduction of enzyme activity with p-chloromercuribenzoate and SKF-525A and its recovery by glutathione suggests the involvement of sulfhydryl group(s) in the 12a-hydroxylase. It is to be
256
noted that the effect of p-chloromercuribenzoate and SKF-525 on 120:-hydroxylation of 5a_cholestane-3cu ,7cu-dial is similar to that reported for 7~ -hydroxychol~st-4-en-3-one [ 131. CO had little effect on the reaction. Bjijrkhem and Danielsson [29] have reviewed the low sensitivity of rat liver 12&-hydroxylase toward CO, and reported an inhibition of about 30% in an atmosphere of 40% CO, 4% Nz and 56% N2. Ichikawa and Mason [30] reported approx. 1.5 nmol of P-450/mg protein for rabbit microsomes. Clearly, the role of cytochrome P-450 in 12a-hydroxylation is still unresolved. The activity of hepatic 12cu-hydroxylase after freezing and thawing is significantly decreased after 2-7 weeks storage at -20°C. Shefer et al. [ 311 reported that rat liver microsomes stored at -15°C for 1 month lost 50% of their activity. Omura and Sato [ 321 used rabbit microsomal suspensions stored at 4” C for 2---3 days. Rabbits of different age or sex (Table II) afforded variable activity of microsomal 12cu-hydroxylase, demonstrating that a single microsomal preparation is essential for each set of experiments. Einarsson f13] reported a 40% increase in 12cu-hydroxylation of 7cu-hydroxycholest-4-en-3-one for the adult male rat over the immature rat, and a 50% higher activity in the adult male rat over the adult female rat. Acknowledgements This investigation was supported by U.S. Public Health Service Grant HE-07878. The authors are pleased to acknowledge the expert technical assistance of John Branca, Eric Stephenson and William Frasure. Dr Burton W. No11 kindly provided a sample of [24-l 4 C J allochenodeoxycholic acid. References 1
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