Rapid feedback inhibition of endogenous cholic and chenodeoxycholic acid synthesis by exogenous chenodeoxycholic acid in man

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).