Vol. 64, No. 2, 1975

BIOCHEMICAL

BILE

ACIDS

AND BIOPHYSICAL

SECRETION

BY ISOLATED

RAT

M. S. Anwer, Institut

fiir

AND

and

D.

Toxikologie

Fachbereich der

Hegner und

Pharmazie

Tiermedizin

Universitat

Leiter: Received

SYNTHESIS

HEPATOCYTES

R. Kroker

Pharmakologie,

RESEARCH COMMUNICATIONS

Miinchen

Prof

Dr.

D.

Hegner

24,1975

March

SUMMARY

Bile acids secretion and their distribution were studied in isolated rat hepatocytes. Bile acids secretion was linearly related with time for first three hours of incubation and the net secretion rate was 21. 7 + 2. 74 nmoles per g cells ( wet weight) per minute. Isolatedhepatocytes synthesized relatively more chenodeoxycholic acid than cholic acid compared to whole animal. These results suggest that isolated hepatocytes synthesize and secrete bile acids and thus provide experimental system to study the effect of drugs on bile acids secretion and synthesis at cellular level.

INTRODUCTION Liver

plays

a major

and excretion have

been

toxic

agent

of bile

of bile Isolated system diseases this

Copyright All rights

acids.

demonstrated

have

been

of liver acids

possiblity,

used

the effect

animal.

acids (1)

(4, 5). However, has not been

and hepatic

bile

acid

secretion

synthetic

reported.

drugs

level.

isolated

metabolism

experimental

at cellular

603

and

a suitable

of different

studied

metabolism

Recently

level

provide

conjugation

and by hepato-

the metabolic

metabolism

we have

D 19 75 by Academic Press. Inc. of reproduction in any form reserved.

diseases

hepatocytes could

acid

in bile

synthesis,

to study

at cellular

hepatocytes

on bile

Changes

(3) in whole

in isolated

to study

in the uptake,

in hepatic

(2) and drugs

hepatocytes activities

role

To evaluate and their

Vol. 64, No. 2, 1975

BIOCHEMICAL

distrubution those

in isolated

of whole

rat

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hepatocytes

diet five

Wistar

with

rats

water

minutes

were

after

duct.

Rat liver

cells

were

and

used

a calcium

(6).

free

0,2%

trypan

(LDH)

(wet

activity

cells

per

Eight

The

cells

to ten ml cell

per

containing

.?“r, bovine

excluding glucose hour

The

20 minutes

0, 1% hyaluronidase was

Average

then

cell

continued

yield

was

and 93 to 96% of the cells

of 125 I.rMoles

was

lactic

one

excluded

dehydrogenase

cell

assayed

albumin

of O2 per

g

with

the dye was

bile

fraction

suspension

trypan

- 160

buffer V and

(pH 7, 4)

1 mg%

by a slow

of preincubation,

at different

time

acids.

At intervals,

an aliquot

blue

and the percent

of cells

determined. were

incubated

(120

in Tyrode

10 min

was

shaking

maintained

After

for bile

100 mg/ml) with

4 cm)

and pH were

from

stained

(ca.

a: 37’C

serum

aeration

were

incubation

determined

for

cells

(7).

of the method

at a rate

amplitude

in the supernatants

Total

of liver

suspension

min,

the supernatants

of cells

perfused

for

and cannulating

a modification

their

of 95% O2 - 5% CO2.

intervals

ether

retained

flasks

oscillation

stream

collected

hour.

in 50 ml Erlenmayer

glucose.

on standard

was

containing

liver

respired

Bile with

was

by Baur per

and

liver

Isolation

weight)

blue.

using

solution

as described

cells

studies. the rats

The

Hank’s

0, 05% collagenase.

mechanically

in all

prepared

and Friend

gram

with

METHODS

250 to 350 g maintained

anesthetizing

bile

with

AND

weighing

the common

of Berry

them

animal.

MATERIALS Male

and compared

Lactate, determined

pyruvate

and

at the end of four-

periods.

acids

in the supernatants

enzymatically

with

and bile

hydroxysteroid

604

sampes

were

dehydrogenase

BIOCHEMICAL

Vol. 64, No. 2,1975

(Grade

II,

Sigma)

Yamasaki The

(8).

taining

by a modification

Bile

reaction

samples

mixture

EDTA

of hydroxy

steroid

at room

temperature

measured

at

nm against

minations

were

366

always

assayed

acids

concentration

coefficient The

solution

performed with

appropriate

of NADH

coefficient

0,02 was

and the extinction All

deter-

A set of standards

from

The

the molar

at 866 nm and net increase

of variation

and

mixture

set of determination.

was calculated

/ml)

blanks,

in duplicates.

each

0, 05 ml

reaction

one hour

con-

(0, 4

(5 units

The

for

buffer

sulfate

solution

and

water.

(10 nmoles/l),

or 0, 1 ml supernatants.

incubated

1M glycine

and hydrazine

dehydrogenase

of Iwata

(1: 10) with

1,O ml

/I)

0, 05 ml of l3-NAD

bile

diluted

contained

(5. 6mmoles

RESEARCH COMMUNICATIONS

of the method

were

moles/l),

diluted

AND BIOPHYSICAL

of dublicate

total

was bile

extrinction

in extinction.

determinations

was

2-48. Individual were

also

bile

one ml

of incubation

(11).

bile

by glucose

oxidase

glucose difference

ger,

were

four

of four

hour

hour

and pyruvate Mannheim

hours

(12).

were

using were

Recoveries

layer

chro-

(9).

Bile

four

hours

The

hour

solvent

system

detected

in

of standard

Glucose

incubation

and zero

was

determined

net production

of

was

as the

calculated

glucose

determined

concentrations.

by the method

of Boehrin-

Ltd.

RESULTS

The

after

acids

92 -9807.

method

thin

in o, 1 ml methanol

bile

or bile.

acids

during

obtained

separation

No free

supernatants

gel G plates

extracted

the supernatants

conjugated

Lactate

finally

chromatographic

II of Hoffmann

and after

on silian

supernatant

were

(10) before

samples

enzymatically

separation

from

either

in bile

determined

matographic acids

acids

intergrity of incubation

AND

DISCUSSION ---

and the metabolic were

state

evaluated

of the cells

by trypan

605

blue

after

exclusion,

four

ml

Vol. 64, No. 2, 1975

BIOCHEMICAL

lactate

pyruvate

ratio,

of LDH

activity.

Trypan

cells.

Lactate

production

I.tmoles

g cells

per

the total living

LDH

was

ratio

was

rate per

activity.

liver

cells

incubation No bile

acid

was

The

Thus,

cells

cubation acids

would

acids

were

appeared (trypan

That

after

released

four

exclusion)

were

one hour

1:

Time

at 37’C

and pH 7, 4. Each

and the vertical were

detected

samples

course

of bile

acids

value

and not

by the following in the inFree

bile if bile

b) Bile

180

acids

damage

of incubation.

c) Bile

240

TIME Imid

secretion

represents

standard

and three

out of five

by rat

mean error

represents

606

1). 10

secreted

represents

weight

after

of no cell

line

Cell

(Fig.

cells.

in two

respectively.

period taken

120

INCUBATION

of incubation.

in the

in the media

inspite

60

to be

acids

of incubation.

media

90% of

hours

detected

the damaged

after

Figure

four

were

to be present

from

retained

support

acids

hours

of the

considered

bile

acids was

bile

in the incubation blue

were

samples

bile

cells

be expected

cells

during

in the

a) No free media

by 80-85”

the incubation

by the damaged

observation,

liver

to secrete

detected

and retention

9, 3 -+ 2, 58 (mean + S. E. M. ) 16 + 4, 1 (mean -+ S. E. M. )

hour.

throughout

RESEARCH COMMUNICATIONS

rate

excluded

functional

of preincubation.

released

was

continued

media

production

blue

and metabolically

The

min

glucose

pyruvate

and glucose

AND BIOPHYSICAL

of five of mean.

studies

hepatocytes studies Bile

acids

in 15 and 30 min

wet weight

BIOCHEMICAL

Vol. 64, No. 2, 1975

acids

could

LDH

activity)

not be detected obtained

freezing

and thawing.

linearly

related

values

were

actually bile

acids The

acids

time

for

be taken

was

(51, 8 nmoles/g

liver/min)

secretion

rate

uptake

acids

Liver

cells

take

(unpublished The

significantly

The

ratio

up as much

in the bile

different

than

the availablity

This was

for

Wistar

rats

male

(9).

hepatocytes

when

are

may

the simultaneous

taken

into

bile

consideration.

acid

in 15 min

acids

acids

precursor

be mostly

A combination

acids

(14).

Since

primary

higher

of an inhibition

bile

than

change

disorder

in conjugation in taurine

during

of glycine

conjugation,

higher

of primary

acids,

Biosynthesis

acids

synthesis

of 12 a-hydroxylase

acid

bile

and not secre-

has been

the bile

to

in the incubation

synthesis

synthesis,

the isolation

of dihydroxy

proportion acid

glycine

resulting

ratio

by

cells.

alteration

The

1).

higher

and as a result

that of cholic

607

(table

may be explained

by the hepatocytes.

of bile

rat hepatocytes

bile

acids

in the liver

significantly

to indicate

media

significantly

This

out.

in the relative

bile

was

Similar

illeal

be ruled

difference

relatively

acids.

also

bile

in the cells

with

was

in the incubation

conjugation

in the preference

considered

would

acids

a metabolic

cannot

of preexisting

was

acids

media.

supply

However,

bile

in isolated

acid

of bile

conjugates

for

in patients

unlikely,

cholesterol,

media

+ 2, 74 nmoles/

excretion

that of biliary

the bile

resulting

trihydroxy

tion

at the same

(23,2

isolated

bile

from

be in short

(13).

procedure

acids

acids

cells

in the incubation

is demonstrated

media.

cells

range

to glycine

to conjugate

although

is

of the secreted

as ZOO7 of added

of substrate

may

depletion

part

by

of individual

of taurine

used

which

data).

was

was

rate

by the liver

distribution

Taurine

the secretion

acids

in the physiological

of bile

three-hour

biliary

be considered

to be

Thus

reported

of bile

by repeated

hours.

of bile

than

to assay

appeared

by the liver

rate

lower

secretion

since

up

(used

the cells

three

rate

net secretion

g cells/min)

The

Bile

to calculate

would

lysates

by disrupting

with

used

in the cell

the net secretion

time.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

demonstrated

in the incubation of chenodeoxycholic

in the hepatocytes. and a stimulation

Vol. 64, No. 2, 1975

Table

BIOCHEMICAL

1: Bile

-

acid

distribution

in bile

--

No.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and isolated

rat

hepatocytes.

------

-

of animals/trials

Taurocholic

acid

(olO)b

Glycocholic

acid

(%)

Bile

Hepatocytes

4(8)a

5(10)

58, 4 + 3, 51’

12, 6 + 1, 66

6, 1 +- 0, 94

23, 0 + 2, 36

Tauro-dihydroxy

bile

acids

(‘$10) 28, 8 -+ 1, 79

43,2

-+ 1,81

Glyco-dihydroxy

bile

acids

(“lo)

4, 2 _+ 0, 95

10,9

-+ 1,63

acid

(q)

2, 7 -+ 0, 69

10,4

+- 1,61

Unidentified

bile

Dihydroxy/trihydroxy

bile

Taurine/glycine

aNo.

All

acids

conjugates

bile

were

b

of microsomal primary would

acids

be expected

ratio

hormone

may

present

synthesize

acid study

and secrete

‘Mean

rate

of any thyroid

for

for

similar

(15).

The

hormone

that

factor

relatively

(s)

level.

increase liver

other

higher

that

bile

acids

isolated and thus

608

rat

hepatocytes

provide

a suitable

in

cells

following

in the hepatocytes.

showed

+ S. E. M.

between

at 95% significance

in hyperthyroid

be responsible

acids,

compared

suggested

it is likely

1, 7 -+ 0, 15

bile

when

t-test

was

Thus,

of chenodeoxycholic The

student’s

to be devoid

procedure.

thyroid

different

26-hydroxylase

bile

isolation

by

of total

Percent

significantly

and hepatocytes

1, 58 -+O, 150

10, 6 -+ 2, 16

of determinations,

values

0, 53 +- 0, 063

the than

synthesis

BIOCHEMICAL

Vol. 64, No. 2, 1975

experimental hepatic level.

system

diseases

the relative

studying

on bile

acids

higher

synthesis

Relatively

the hepatocytes

for

AND BIOPHYSICAL

could

be used

proportion

the effects

secretion

RESEARCH COMMUNICATIONS

of drugs

and

and synthesis

at cellular

of chenodeoxycholic

to study

of primary

acid

the mechanism

bile

acids

in

controlling

synthesis

from

cholesterol.

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1.

Nair, P. P., and Kritchevsky, D. (1973) pp. 55-82, Plenum Press, New York.

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Anwer, (1975)

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Crane, L. J., Commun. 60,

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Sundler, Chem.

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6.

Berry, 506-520.

M. N.,

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Paumgartner, G., Naunyn-Schmiedeberg’

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K. W., Lack, 56, 71 I-720.

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Nilsson, Biochem.

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