Vol. 64, No. 3,1975
BIOCHEMICAL
RAPID FEEDBACK INHIBITION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF ENDOGENOUS CHOLIC AND CHENODEOXYCHOLIC
ACID SYNTHESIS BY EXOGENOUS CHENODEOXYCHOLIC ACID IN MAN* Leon
Swell,
Charles
C. Schwartz,
L. Gregg
Halloran,
Veterans Administration Virginia Commonwealth Richmond, Virginia Received
April
and Z. Reno Vlahcevic
Hospital University 23249
and
23,197s SUMMARY
Chenodeoxycholic acid (300 mg + 14C) was administered orally to a bile fistula patient receiving a constant infusion of {3H)mevalonic acid. Suppression of endogenous cholic and chenodeoxycholic acid synthesis occurred within 2 to 4 hours and continued for the next 10 hours; synthesis returned to the baseline level after 18 hours. Incorporation of (3H}mevalonic acid into both bile acids was also greatly reduced during the first several hours after chenodeoxycholic acid, but almost recovered by 5 hours. The data suggest that multiple feedback sites are involved in the regulation of bile acid synthesis in man.
It
has been
7u-hydroxylase
which
the
7a-hydroxylation
act
to regulate
precursor. reports
limited synthesis
their
with
have
shown that related
information
which
the
conversion are
the isotope gallstones
for
has been
*Sunuorted in part 2Rb'l-AM-14668104, Service.
obtained
Inc. reserved.
that
of cholesterol
chenodeoxycholic
on the effect
inhibits
cholic
of administered
Public Health of Health,
Service United
but recent is
in man.
term It
acid
acids
of bile
long
gallstones.
and
synthesis
regulation the
bile
rat,
synthesis
with
markedly
1083
in the
acid
cholesterol
of HMG-CoA reductase,
cholesterol
to a year
bv United States National Institutes
D 19 75 by Academic Press, of’ reproduction in arz.v form
predominantly
in conjunction
technique
on which
obligatory
on the in vivo
to solubilize
6 months
sites
of their
the regulation
is available
and
of HMG-CoA to mevalonic
the feedback
to the control
dilution
enzymes HMG-CoA reductase
and that
in man have been obtained acid
the
have been made
intimately
No information
Copyright All rig-hts
that
own synthesis
findings
of chenodeoxycholic using
catalyze
(l-6)
of cholesterol
These (7,8)
probably
demonstrated
The
acid
feeding
has been
shown,
fed to patients acid
synthesis
chenodeoxycholic
Research Grant States Public Health
(9)
Vol. 64, No. 3, 1975
acid
on its
measure
BIOCHEMICAL
own synthesis,
endogenous
since
synthesis
In the present
report,
(hours)
of administered
effect
both
cholic
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the isotope
following
data
dilution
the administration
are provided
for
acid
acid
and its
cannot
be used
of the same bile
the first
chenodeoxycholic
and chenodeoxycholic
technique
time
acid.
in man on the immediate
on the endogenous
possible
to
site(s)
synthesis
of
of action.
MATERIAL AND METHODS Patient. T-tube
The patient
inserted
exploration after
in the common duct
for
bile
flow
T-tube
was assured
(16 uCi/hr) for
was open
averaged
and verified
9 hours.
administered
via
chenodeoxycholic
for
7 days prior
the
start
nasogastric
tube
acid.
was collected
Bile
collection
infusion (9 a.m.)
and a common duct
was carried
of a balloon
A constant
after
male who had a Baldwin
out
3 weeks
to the experiment,
Complete
by the inflation
hours
white
The experiment
in the morning
Four
old
cholecystectomy
900 ml/day.
by x-ray.
was started
following
gallstones.
The T-tube
the experiment
rate
cholesterol
surgery.
patient's
used was a 56 year
and the
of bile
in the
during
distal
arm of the
of {5-3H)mevalonic and was maintained
of the infusion,
at 20 minute
at the same
the
300 mg of chenodeoxycholic
acid
acid
patient plus
intervals
was
8 uCi C14C)-
throughout
the
experiment. Labeled
Compounds.
from New England sodium
Nuclear
bicarbonate
solution
Corp.
followed
of sodium
HCl and diluted was passed
The DL-{5-3H)mevalonic and liberated
by ethyl-ether
{3H}mevalonate
with
through
sterile
saline
filter
Inc.
and checked
for
using
a solvent
system
The chloroform and for
phase
cholesterol
of isooctane-ethyl
was analyzed by the method
with for
with
an equimolar
20 volumes phospholipid
1084
from ICN Pharmaceuticals,
acid
on silica
gel
G
10:10:2.
chloroform
by the method
and Webb (11).
of
intravenously
chromatography
of 2:l
amount
The solution
of 50 ml.
and administered
layer
of
The aqueous
of DBED.
acetate-acetic
of Sperry
was obtained
by the addition
was obtained
(97%) by thin
was extracted
the salt
to a volume
(0.2211)
The {24-14C}chenodeoxycholic
Bile
(DBED salt)
extraction
to the patient.
Methods.
from
was neutralized
normal
a millipore
purity
acid
Bile
methanol. of Bartlett acids
were
(10)
Vol. 64, No. 3,1975
determined
by a combination
described were
BIOCHEMICAL
earlier
effect
for
quench
a) bile
acid
studied
had a constant
the effect
possible
the secretion bile
acid not
synthesis a bile
in the bile
acids
into
cholesterol
rate
limiting
activity the
into
in
3H into
bile
cholesterol
of action
should steps both
not
step
acids in
could
acid.
from
the buildup
infusion
therefore
the bile
acid
acid
indicate
there
effect
the
specific
does
an evaluation limiting
of 3H activity
is no evidence
depression
for
of radio-
of a suppression
Alternatively,
relative
a greated
for
The incorporation
pathway.
into
of 3H activity
on the rate
be indicative
synthesized
acid
patient
allowed acid
since
and
chenodeoxycholic fistula
Significant
could
synthesis
of known
the bile
synthesis.
mevalonic
the mass of bile acids
acid
have been effected
beyond bile
acid
and
simultaneously
acid
of chenodeoxycholic
bile
acid
of endogenous
in this
state,
of {3H}mevalonic
deviation
{3H}mevalonic
since
a steady bile
the
b) the patient
by determining
since
Significant
and/or
7u-hydroxylation
decrease
pool.
studying
synthesis,
implying
incorporation
dilution
Analytic)
synthesis
on endogenous
determination
fractions
(Searle
of {14C}chenodeoxycholic
isotope
site(s)
acid
acid
rate
acid
was used for
bile
to bile
the bile
as
method.
patient
acids
and the
following
of cholesterol
standard
be ascertained
the direct
acid
of the possible steps
could
by simple
counter
secretion
bile
acids
for
scintillation
equal
acid
The administration
allowed
have
is
bile
site(s)
of
on endogenous
secretion
of bile
acids.
activity
acid
chromatography
% activities
The T-tube
of administered
feedback
and gas liquid
external
of the Experiment.
model:
c)
liquid
by the
of chenodeoxycholic
layer
l4 C and
The
in a Mark III
and corrected Design
of thin
(12).
determined
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of
a greater
to the incorporation of chenodeoxycholic
of acid
on
synthesis. RESULTS
Fig. acid
1 shows
The secretion
synthesis.
constant deoxycholic
the effect
prior
of the of cholic
to the administration acid
rapidly
chenodeoxycholic
appeared
1085
on endogenous
and chenodeoxycholic
of chenodeoxycholic in bile
acid
and reached
acids acid.
a peak in
bile
was relatively
Exogenous 2 hours
chenoand then
Vol. 64, No. 3, 1975
BIOCHEMICAL
0
2
4(O)
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2
4
6 8 HOURS
IO
12
14
16
18
of exogenous chenodeoxycholic acid (CDCA) on the secretion Fig. 1. Effect of endogenous bile acids. O-0 = cholic acid (endogenous) X-X = chenodeoxycholic acid (endogenous Ad = chenodeoxycholic acid (exogenous).
declined.
Recovery
in 8 hours
and 92% for
deoxycholic
acid
within
acid
14th hour
of both
bile
14 hour
period
cholic were
and then
found
mevalonic a plateau.
into acid
infusion
Within
was a marked
40% for
cholic
so that
to the original
return
acid,
of
acid
the average
During
decrease
bile of
3H activity
following 3H activity
chenodeoxycholic of 3H incorporation
1086
acid. into
period.
4 hours acids
administration
into
decreases
on the incorporation
in bile
the
the 4 to
in endogenous These
Following
acids.
both
both
tended
of of 13H}to reach
of chenodeoxycholic
bile
During
until
the synthesis
to the control
acid
of cheno-
continued
18th hour level.
after
was inhibited
synthesis
by the
when compared
4 hours
inhibition
was 32% and 29% respectively.
synthesized the output
first
in the
was 70% and cheno-
synthesis
baseline
of chenodeoxycholic
1.5 to 2 hours
and 60% for
was a gradual
to increase
(PcO.01)
depression
delay
activity cholic
the
acid
of bile
acids
the newly
cholic
The depression
chenodeoxycholic
2 shows the effect
within
was a 1.5 to 2 hour
started
to be significant
suppressed While
acid
Endogenous
period.
rapidly
had returned
after
3H activity
there
there
synthesis.
acids
18 hour
were
and chenodeoxycholic
Fig.
there
total
administration.
1.5 hours,
deoxycholic the
acid
1 to
the
synthesis
chenodeoxycholic
{ 14C)chenodeoxycholic
of administered
acids
which
the next bile
acids.
averaged
3.5 hours
BlOCHEMICAL
Vol. 64, No. 3,1975
6000
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t
r 2 4000 5 ; I b
2000
0
I
2
3
40) I HOURS
2
3
4
5
Fig. 2. Effect of exogenous chenodeoxycholic acid (CDCA) on the incorporation Values for cholic acid were corrected of {3H)mevalonic acid into bile acids. for random loss (8.33%) of 3H activity at position 12 of cholesterol during 0-O = cholic acid X-X = chenodeoxycholic acid. conversion to bile acids.
Fig.
3 shows the effect
of chenodeoxycholic
cholesterol,
phospholipid,
and total
lipids
was relatively
constant
acid.
The total
acid
deoxycholic
bile
acid
paralleled
the
followed total
bile
prior
peaked
acids.
to the
rise
but
salts,
on the secretion
The secretion
administration
at 2 hours
by a second
bile
acid
after
of the biliary
of chenodeoxycholic
the administration
at 6 hours.
there
of biliary
of cheno-
Phospholipid
was no appreciable
secretion
change
in cholesterol
secretion. DISCUSSION The data dose
of the present
report
have
(300 mg) of chenodeoxycholic
synthesis
of both
suppressive period
suggests
common (at The initial state of bile
effect
cholic
and the continued
rapid
in part) phase
to the pathway
of the enzyme protein acid
administered
synthesis
inhibition
acid
chenodeoxycholic
of both
acid
type
4 hours
had been
1087
acids
site(s)
cholic is
bile
over
a 10 hour which
and chenodeoxycholic with
of feedback
eliminated
of this
in the liver
consistent
even
of a small
the endogenous
The rapidity
feedback
inhibition
after
inhibited
of both
on an allosteric
continued
the administration
acids.
of one or more
of bile
that
to man markedly
and chenodeoxycholic
the presence
least
acid
shown
though
a change
in the bile.
acids. in the Inhibition
inhibition.
a major
is
portion Since
of the f3Hl
Vol. 64, No. 3,1975
Fig. &-C
BIOCHEMICAL
3. Effect of chenodeoxycholic acid = phospholipid X-X = cholesterol
mevalonic
acid
administered
incorporation
into
chenodeoxycholic
hydroxylation
step
of the endogenous
However,
in the
later
phase
of 3H radioactivity
inhibition
of bile
the bile then
acid
fraction
which
the bile
synthesis.
bile
acids
the dissociation
between
incorporation
of
acid
This
inhibition
resulted acid
{3H)mevalonic
in a decreased
synthesis
occur
amount
of higher
of endogenous
bile
have
endogenous
at the level of newly
of 3H activity
acid
synthesis,
cholesterol after
most
1088
acid
acids
synthesis
suggests
feedback
of 7a-hydroxylase to hydroxylate
substrate. of the
the
would
have
fOK bile
fraction
could
activity
since
the smaller
The prolonged
exogenous
after
was effected. which
acid
fashion.
and the
that
site
in
activity,
in a parallel
in the bile
be sufficient
activity
increased
cholesterol
of enzyme would
of incorpora-
of 3H activity
synthesized
inhibition
2 hours.
was a continuous
of HMG-CoA reductase
of a continuous
specific
there
7a-
the reduced
of 7a-hydroxylase
bile
bile
for
the first
recovery
an additional
availability Recovery
(13).
in the presence
even a reduced amount
have been
even though
by the of the
was a recovery
by a return
both
inhibition
during
there
secretion.
effected
responsible
occurred
should
into
of 7a-hydroxylase
could
partly
lipid acids.
not
that
the observed
caused
Consequently,
suppression
is
acids If
was solely
the mass of endogenous
initial
seems likely
pathway
acids
is probably
of the experiment,
into
acids
it
acid
bile
(CDCA) on biliary A-4 = total bile
cholesterol
acid,
in the bile
labeling
tion
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
suppression
chenodeoxycholic
Vol. 64, No. 3,1975
acid
BIOCHEMICAL
had been eliminated
secondary
feedback
in the bile
effect
enzyme HMG-CoA reductase chenodeoxycholic since
cholic
chenodeoxycholic
is
secretion significantly
acid
delay
with
a reduction
also
suggest
The data feedback
inhibition
was suppressed
initially
of chenodeoxycholic
the earlier closely
consistent
(14).
synthesis
and the time
necessary in
to invoke
the synthesis
that
the mechanism
may not
be entirely
to a greater
extent
a of the
of similar than
acid.
The effect with
are
and cholic acid
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
findings
linked
to the
(15) influx
is more independent altered
acid
on biliary
in man and of bile of changes
by chenodeoxycholic
lipid
indicates
acids
into
in bile
secretion
that the acid
is
phospholipid
liver. concentration
consistent secretion
Cholesterol and was not
acid.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Siperstein, M.D., and Fagan, V.J., J. Biol Chem. 241, 602 (1966). Shefer, S., Hauser, S., Bekersky, I., and Mosbach, E.H., J. Lipid Res. 11, 404 (1970). Mosbach, E.H., Arch. Intern. Med. m, 477 (1972). McNamara, D.J. and Rodwell, V.W., In Biochemical Regulatory Mechanisms in the Eucaryotic Cell, 205-242, 1972TJohn Wiley and Sons, New York, N.Y. Hampretch, B., Niissler, C., Waltinger, G., and Lynen, F., Eur. J. Biochem. 2, 10 (1971). Shapiro, D.J. and Rodwell, V.W., J. Biol. Chem. 46, 3210 (1971). Goldstein, J.L. and Brown, M.S., Proc. Nat. Acad. Sci. 70, 2804 (1973). Fogelman, A.M., Edmond, J., Seager, J., and Popjak G., J. Biol. Chem. 250, 2045 (1975). Danzinger, R.G., Hofmann, A.F., Thistle, J.L., and Schoenfield, L.J., J. Clin. Invest. 52, 2809 (1973). Bartlett, G.R., J. Biol. Chem. 234, 466 (1959). Sperry, W.M. and Webb, J., J. Biol. Chem. 187, 97 (1950). Vlahcevic, Z.R., Bell, C.C., Jr., Buhac, I., Farrar, J.T., and Swell, L., Gastro. 2, 165 (1970). Balasubramaniam, S., Mitropoulos, K.A., Myant, N.B., Eur. J. Biochem. 2, 77 (1973). Higgins, M.J.P., Brady, D., and Rudney, H., Arch. Biochem. Biophys. 163, 271 (1974). Bell, C.C., Jr., Vlahcevic, Z.R., and Swell, L., Surg. Gyn. and Obst., 132, 36 (1971).
BIOCHEMICAL
RAPID FEEDBACK INHIBITION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF ENDOGENOUS CHOLIC AND CHENODEOXYCHOLIC
ACID SYNTHESIS BY EXOGENOUS CHENODEOXYCHOLIC ACID IN MAN* Leon
Swell,
Charles
C. Schwartz,
L. Gregg
Halloran,
Veterans Administration Virginia Commonwealth Richmond, Virginia Received
April
and Z. Reno Vlahcevic
Hospital University 23249
and
23,197s SUMMARY
Chenodeoxycholic acid (300 mg + 14C) was administered orally to a bile fistula patient receiving a constant infusion of {3H)mevalonic acid. Suppression of endogenous cholic and chenodeoxycholic acid synthesis occurred within 2 to 4 hours and continued for the next 10 hours; synthesis returned to the baseline level after 18 hours. Incorporation of (3H}mevalonic acid into both bile acids was also greatly reduced during the first several hours after chenodeoxycholic acid, but almost recovered by 5 hours. The data suggest that multiple feedback sites are involved in the regulation of bile acid synthesis in man.
It
has been
7u-hydroxylase
which
the
7a-hydroxylation
act
to regulate
precursor. reports
limited synthesis
their
with
have
shown that related
information
which
the
conversion are
the isotope gallstones
for
has been
*Sunuorted in part 2Rb'l-AM-14668104, Service.
obtained
Inc. reserved.
that
of cholesterol
chenodeoxycholic
on the effect
inhibits
cholic
of administered
Public Health of Health,
Service United
but recent is
in man.
term It
acid
acids
of bile
long
gallstones.
and
synthesis
regulation the
bile
rat,
synthesis
with
markedly
1083
in the
acid
cholesterol
of HMG-CoA reductase,
cholesterol
to a year
bv United States National Institutes
D 19 75 by Academic Press, of’ reproduction in arz.v form
predominantly
in conjunction
technique
on which
obligatory
on the in vivo
to solubilize
6 months
sites
of their
the regulation
is available
and
of HMG-CoA to mevalonic
the feedback
to the control
dilution
enzymes HMG-CoA reductase
and that
in man have been obtained acid
the
have been made
intimately
No information
Copyright All rig-hts
that
own synthesis
findings
of chenodeoxycholic using
catalyze
(l-6)
of cholesterol
These (7,8)
probably
demonstrated
The
acid
feeding
has been
shown,
fed to patients acid
synthesis
chenodeoxycholic
Research Grant States Public Health
(9)
Vol. 64, No. 3, 1975
acid
on its
measure
BIOCHEMICAL
own synthesis,
endogenous
since
synthesis
In the present
report,
(hours)
of administered
effect
both
cholic
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the isotope
following
data
dilution
the administration
are provided
for
acid
acid
and its
cannot
be used
of the same bile
the first
chenodeoxycholic
and chenodeoxycholic
technique
time
acid.
in man on the immediate
on the endogenous
possible
to
site(s)
synthesis
of
of action.
MATERIAL AND METHODS Patient. T-tube
The patient
inserted
exploration after
in the common duct
for
bile
flow
T-tube
was assured
(16 uCi/hr) for
was open
averaged
and verified
9 hours.
administered
via
chenodeoxycholic
for
7 days prior
the
start
nasogastric
tube
acid.
was collected
Bile
collection
infusion (9 a.m.)
and a common duct
was carried
of a balloon
A constant
after
male who had a Baldwin
out
3 weeks
to the experiment,
Complete
by the inflation
hours
white
The experiment
in the morning
Four
old
cholecystectomy
900 ml/day.
by x-ray.
was started
following
gallstones.
The T-tube
the experiment
rate
cholesterol
surgery.
patient's
used was a 56 year
and the
of bile
in the
during
distal
arm of the
of {5-3H)mevalonic and was maintained
of the infusion,
at 20 minute
at the same
the
300 mg of chenodeoxycholic
acid
acid
patient plus
intervals
was
8 uCi C14C)-
throughout
the
experiment. Labeled
Compounds.
from New England sodium
Nuclear
bicarbonate
solution
Corp.
followed
of sodium
HCl and diluted was passed
The DL-{5-3H)mevalonic and liberated
by ethyl-ether
{3H}mevalonate
with
through
sterile
saline
filter
Inc.
and checked
for
using
a solvent
system
The chloroform and for
phase
cholesterol
of isooctane-ethyl
was analyzed by the method
with for
with
an equimolar
20 volumes phospholipid
1084
from ICN Pharmaceuticals,
acid
on silica
gel
G
10:10:2.
chloroform
by the method
and Webb (11).
of
intravenously
chromatography
of 2:l
amount
The solution
of 50 ml.
and administered
layer
of
The aqueous
of DBED.
acetate-acetic
of Sperry
was obtained
by the addition
was obtained
(97%) by thin
was extracted
the salt
to a volume
(0.2211)
The {24-14C}chenodeoxycholic
Bile
(DBED salt)
extraction
to the patient.
Methods.
from
was neutralized
normal
a millipore
purity
acid
Bile
methanol. of Bartlett acids
were
(10)
Vol. 64, No. 3,1975
determined
by a combination
described were
BIOCHEMICAL
earlier
effect
for
quench
a) bile
acid
studied
had a constant
the effect
possible
the secretion bile
acid not
synthesis a bile
in the bile
acids
into
cholesterol
rate
limiting
activity the
into
in
3H into
bile
cholesterol
of action
should steps both
not
step
acids in
could
acid.
from
the buildup
infusion
therefore
the bile
acid
acid
indicate
there
effect
the
specific
does
an evaluation limiting
of 3H activity
is no evidence
depression
for
of radio-
of a suppression
Alternatively,
relative
a greated
for
The incorporation
pathway.
into
of 3H activity
on the rate
be indicative
synthesized
acid
patient
allowed acid
since
and
chenodeoxycholic fistula
Significant
could
synthesis
of known
the bile
synthesis.
mevalonic
the mass of bile acids
acid
have been effected
beyond bile
acid
and
simultaneously
acid
of chenodeoxycholic
bile
acid
of endogenous
in this
state,
of {3H}mevalonic
deviation
{3H}mevalonic
since
a steady bile
the
b) the patient
by determining
since
Significant
and/or
7u-hydroxylation
decrease
pool.
studying
synthesis,
implying
incorporation
dilution
Analytic)
synthesis
on endogenous
determination
fractions
(Searle
of {14C}chenodeoxycholic
isotope
site(s)
acid
acid
rate
acid
was used for
bile
to bile
the bile
as
method.
patient
acids
and the
following
of cholesterol
standard
be ascertained
the direct
acid
of the possible steps
could
by simple
counter
secretion
bile
acids
for
scintillation
equal
acid
The administration
allowed
have
is
bile
site(s)
of
on endogenous
secretion
of bile
acids.
activity
acid
chromatography
% activities
The T-tube
of administered
feedback
and gas liquid
external
of the Experiment.
model:
c)
liquid
by the
of chenodeoxycholic
layer
l4 C and
The
in a Mark III
and corrected Design
of thin
(12).
determined
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of
a greater
to the incorporation of chenodeoxycholic
of acid
on
synthesis. RESULTS
Fig. acid
1 shows
The secretion
synthesis.
constant deoxycholic
the effect
prior
of the of cholic
to the administration acid
rapidly
chenodeoxycholic
appeared
1085
on endogenous
and chenodeoxycholic
of chenodeoxycholic in bile
acid
and reached
acids acid.
a peak in
bile
was relatively
Exogenous 2 hours
chenoand then
Vol. 64, No. 3, 1975
BIOCHEMICAL
0
2
4(O)
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2
4
6 8 HOURS
IO
12
14
16
18
of exogenous chenodeoxycholic acid (CDCA) on the secretion Fig. 1. Effect of endogenous bile acids. O-0 = cholic acid (endogenous) X-X = chenodeoxycholic acid (endogenous Ad = chenodeoxycholic acid (exogenous).
declined.
Recovery
in 8 hours
and 92% for
deoxycholic
acid
within
acid
14th hour
of both
bile
14 hour
period
cholic were
and then
found
mevalonic a plateau.
into acid
infusion
Within
was a marked
40% for
cholic
so that
to the original
return
acid,
of
acid
the average
During
decrease
bile of
3H activity
following 3H activity
chenodeoxycholic of 3H incorporation
1086
acid. into
period.
4 hours acids
administration
into
decreases
on the incorporation
in bile
the
the 4 to
in endogenous These
Following
acids.
both
both
tended
of of 13H}to reach
of chenodeoxycholic
bile
During
until
the synthesis
to the control
acid
of cheno-
continued
18th hour level.
after
was inhibited
synthesis
by the
when compared
4 hours
inhibition
was 32% and 29% respectively.
synthesized the output
first
in the
was 70% and cheno-
synthesis
baseline
of chenodeoxycholic
1.5 to 2 hours
and 60% for
was a gradual
to increase
(PcO.01)
depression
delay
activity cholic
the
acid
of bile
acids
the newly
cholic
The depression
chenodeoxycholic
2 shows the effect
within
was a 1.5 to 2 hour
started
to be significant
suppressed While
acid
Endogenous
period.
rapidly
had returned
after
3H activity
there
there
synthesis.
acids
18 hour
were
and chenodeoxycholic
Fig.
there
total
administration.
1.5 hours,
deoxycholic the
acid
1 to
the
synthesis
chenodeoxycholic
{ 14C)chenodeoxycholic
of administered
acids
which
the next bile
acids.
averaged
3.5 hours
BlOCHEMICAL
Vol. 64, No. 3,1975
6000
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
t
r 2 4000 5 ; I b
2000
0
I
2
3
40) I HOURS
2
3
4
5
Fig. 2. Effect of exogenous chenodeoxycholic acid (CDCA) on the incorporation Values for cholic acid were corrected of {3H)mevalonic acid into bile acids. for random loss (8.33%) of 3H activity at position 12 of cholesterol during 0-O = cholic acid X-X = chenodeoxycholic acid. conversion to bile acids.
Fig.
3 shows the effect
of chenodeoxycholic
cholesterol,
phospholipid,
and total
lipids
was relatively
constant
acid.
The total
acid
deoxycholic
bile
acid
paralleled
the
followed total
bile
prior
peaked
acids.
to the
rise
but
salts,
on the secretion
The secretion
administration
at 2 hours
by a second
bile
acid
after
of the biliary
of chenodeoxycholic
the administration
at 6 hours.
there
of biliary
of cheno-
Phospholipid
was no appreciable
secretion
change
in cholesterol
secretion. DISCUSSION The data dose
of the present
report
have
(300 mg) of chenodeoxycholic
synthesis
of both
suppressive period
suggests
common (at The initial state of bile
effect
cholic
and the continued
rapid
in part) phase
to the pathway
of the enzyme protein acid
administered
synthesis
inhibition
acid
chenodeoxycholic
of both
acid
type
4 hours
had been
1087
acids
site(s)
cholic is
bile
over
a 10 hour which
and chenodeoxycholic with
of feedback
eliminated
of this
in the liver
consistent
even
of a small
the endogenous
The rapidity
feedback
inhibition
after
inhibited
of both
on an allosteric
continued
the administration
acids.
of one or more
of bile
that
to man markedly
and chenodeoxycholic
the presence
least
acid
shown
though
a change
in the bile.
acids. in the Inhibition
inhibition.
a major
is
portion Since
of the f3Hl
Vol. 64, No. 3,1975
Fig. &-C
BIOCHEMICAL
3. Effect of chenodeoxycholic acid = phospholipid X-X = cholesterol
mevalonic
acid
administered
incorporation
into
chenodeoxycholic
hydroxylation
step
of the endogenous
However,
in the
later
phase
of 3H radioactivity
inhibition
of bile
the bile then
acid
fraction
which
the bile
synthesis.
bile
acids
the dissociation
between
incorporation
of
acid
This
inhibition
resulted acid
{3H)mevalonic
in a decreased
synthesis
occur
amount
of higher
of endogenous
bile
have
endogenous
at the level of newly
of 3H activity
acid
synthesis,
cholesterol after
most
1088
acid
acids
synthesis
suggests
feedback
of 7a-hydroxylase to hydroxylate
substrate. of the
the
would
have
fOK bile
fraction
could
activity
since
the smaller
The prolonged
exogenous
after
was effected. which
acid
fashion.
and the
that
site
in
activity,
in a parallel
in the bile
be sufficient
activity
increased
cholesterol
of enzyme would
of incorpora-
of 3H activity
synthesized
inhibition
2 hours.
was a continuous
of HMG-CoA reductase
of a continuous
specific
there
7a-
the reduced
of 7a-hydroxylase
bile
bile
for
the first
recovery
an additional
availability Recovery
(13).
in the presence
even a reduced amount
have been
even though
by the of the
was a recovery
by a return
both
inhibition
during
there
secretion.
effected
responsible
occurred
should
into
of 7a-hydroxylase
could
partly
lipid acids.
not
that
the observed
caused
Consequently,
suppression
is
acids If
was solely
the mass of endogenous
initial
seems likely
pathway
acids
is probably
of the experiment,
into
acids
it
acid
bile
(CDCA) on biliary A-4 = total bile
cholesterol
acid,
in the bile
labeling
tion
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
suppression
chenodeoxycholic
Vol. 64, No. 3,1975
acid
BIOCHEMICAL
had been eliminated
secondary
feedback
in the bile
effect
enzyme HMG-CoA reductase chenodeoxycholic since
cholic
chenodeoxycholic
is
secretion significantly
acid
delay
with
a reduction
also
suggest
The data feedback
inhibition
was suppressed
initially
of chenodeoxycholic
the earlier closely
consistent
(14).
synthesis
and the time
necessary in
to invoke
the synthesis
that
the mechanism
may not
be entirely
to a greater
extent
a of the
of similar than
acid.
The effect with
are
and cholic acid
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
findings
linked
to the
(15) influx
is more independent altered
acid
on biliary
in man and of bile of changes
by chenodeoxycholic
lipid
indicates
acids
into
in bile
secretion
that the acid
is
phospholipid
liver. concentration
consistent secretion
Cholesterol and was not
acid.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Siperstein, M.D., and Fagan, V.J., J. Biol Chem. 241, 602 (1966). Shefer, S., Hauser, S., Bekersky, I., and Mosbach, E.H., J. Lipid Res. 11, 404 (1970). Mosbach, E.H., Arch. Intern. Med. m, 477 (1972). McNamara, D.J. and Rodwell, V.W., In Biochemical Regulatory Mechanisms in the Eucaryotic Cell, 205-242, 1972TJohn Wiley and Sons, New York, N.Y. Hampretch, B., Niissler, C., Waltinger, G., and Lynen, F., Eur. J. Biochem. 2, 10 (1971). Shapiro, D.J. and Rodwell, V.W., J. Biol. Chem. 46, 3210 (1971). Goldstein, J.L. and Brown, M.S., Proc. Nat. Acad. Sci. 70, 2804 (1973). Fogelman, A.M., Edmond, J., Seager, J., and Popjak G., J. Biol. Chem. 250, 2045 (1975). Danzinger, R.G., Hofmann, A.F., Thistle, J.L., and Schoenfield, L.J., J. Clin. Invest. 52, 2809 (1973). Bartlett, G.R., J. Biol. Chem. 234, 466 (1959). Sperry, W.M. and Webb, J., J. Biol. Chem. 187, 97 (1950). Vlahcevic, Z.R., Bell, C.C., Jr., Buhac, I., Farrar, J.T., and Swell, L., Gastro. 2, 165 (1970). Balasubramaniam, S., Mitropoulos, K.A., Myant, N.B., Eur. J. Biochem. 2, 77 (1973). Higgins, M.J.P., Brady, D., and Rudney, H., Arch. Biochem. Biophys. 163, 271 (1974). Bell, C.C., Jr., Vlahcevic, Z.R., and Swell, L., Surg. Gyn. and Obst., 132, 36 (1971).
