Suppression by ethanol of acetate and hexanoate oxidation studied by nonrecirculating liver perfusion

Vol. 41, No. 3,197O

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SUPPRESSIONBY ETHANOLOF ACETATE AND HEXANOATEOXIDATION STUDIED BY NONRECIRCULATING LIVER PERFUSION

Kai 0. Lindros Research Laboratories of the State Alcohol Helsinki 10, Finland

Monopoly (Alko),

Received September 25, 1970 SUMMARY The relationship between the effect of ethanol on the redox state and the tricarboxylic acid cycle has been studied by perfuaion of hemoglob n-free mediy$ through the liver without recirculation. The production If co2 from lC-acetate wa6 6UppreEEied 63 % by ethanol and that from Of,4 lC-hexanoate by 54 % in normal livers. These suppressions were much weaker in livers from hyperthyroid rats and stronger in those from hypothyroid rats. Condition6 where the ethanol-induced elevation of the lactate/pyruvate ratio was decreased led to less suppression of substrate oxidation by ethanol. The oxidation of the activity believed extra-

of ethanol

of the citric

and the intramitochondrial

hyperthyroid possibility acid cycle

of a correlation by ethanol

able to follow

production perfusion

previously

output

effects

rat

substrates,

livers

Since previous

(2,4,5),

effect

the

to follow

continuously,

of alterations shift

In order to

of ethanol

on the

the nonrecirculating

used by Exton and Park (6)

possible

the ethanol-induced

(l-4),

redox change is diminished in

and the redox change was investigated.

This method also made it

the metabolic

caused by ethanol

an open question.

the immediate and direct

technique

of both the

between the suppression of the tricarboxylic

of CO2 from different

and acetaldehyde

is still

in hypothyroid

is generally

towards reduction

the ethanol-induced

and increased

This effect

(I-3).

redox state

mechanism of action

f3tUdie6 had shown that

modified

acid cycle

ie known to lead to impairment

to depend in some way on the shift

but the exact

be

in the liver

the rate

wa6 employed.

of ethanol

removal

and to observe on the same liver in the influent of the redox state.

635

medium, which

Vol. 41, No. 3,197O

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIAL AND METHODS Male albino

rals

of the Uistar

animals were fed an ordinary before

use. Induciion

administration as described

and Bi2cher (7).

consisted O/5

technique

The portal

the liver

or acetaldchyde. medium entering a Clark

tension

bicarbonate

mMpyruvate,

if

envelope

or leaving

by

respectively,

to prevent

type oxygen electrode

described

at a rate

11

1.3

The liver

any evaporation

fluid anoxia.

with

mMglucose,

stated.

by Scholz

of 60 ml/min and

equilibrated

not otherwise

the liver.

95 % mW

L-

was en-

of C02, ethanol

in tubes kept at O°C from the The oxygen consumption was measured

(Radiometer), which registered

the oxygen

medium. from 'I5 ml samples after

of the liberated

CO2 in hyanine;

was measured in an EKCOliquid

and pyruvate

was performed

and perfusion

solution

Samples were collected

14 COB was determined

solution

overnight

in order to avoid

and was supplemented with

of the effluent

absorption

was catheterized

medium passed through

0.1

states

from that

in situ

closed in a plastic

with

was modified vein

of Krebs-Ringer

and

and were fasted

and propylthiouracil,

the liver

% CO2 at 37'C

lactate

diet

(4).

pumped through

The perfusion

(300-400 g> were used. The.

and hypothy;-oid

of triiodothyronine

The perfusion

immediately

laboratory

of hyper-

previously

strain

acidification

the radioactivity

scintillation

and of the

counter.

Lactate

(8) withou t deproteinization

were assayed enzymatically

of

the medium.

RESULTS AHD DISCUSSION Physiological

levels

of lactate

added to the medium in order changes induced by the liver. only once through

the liver,

(1.3

to facilitate It

mM) and pyruvate the registration

was found that

the lactate/pvruvate

636

(0.1 mM) were of redox

al.though the fluid ratio

passed

was maintained

:;t

BIOCHEMICAL

Vol. 41, No. 3,197O

Y-10

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

even when moderate alterations

concentration

of acetate

as no alterations

or hexanoate

ously and quantitatively metabolites

was also found to be constant

were made in the entering

changes induced by ethanol

or other

medium (Fig.

metabolites

2-3 minutes after 2,5M

as long

Metabolic

I).

could be followed

method. New steady state

by this

were attained

or pyruvate

medium. The oxygen donsumptjon and the rate

of the influent

of oxidation

were made in the lactate

continu-

output

such an addition

levels

(Fig.

1).

ETH3 PRESENT

80

1000

10

20

30

min.

Fig. I. Demonstration of the rapid adjustment of the steady state levels of the la tate/pyruvate ratios (O-0-0) and the rate of 14C02 formation from I4 C-acetate (O-O-0) in the presence of ethanol in nonrecirculating perfusion of rat liver. Samples were taken f om the medium leaving the liver and assayed for its content of I%0 2' lactate and pyruvate. Addition

of ethanol

pyruvate

ratio

pyruvate

reduced/g

lactate/pyruvate elevation

from about liver ratio

10

to 50-60.

wet wt./min.

perfusion

equivalents

(3,Y).

to 0.45 umol

In the presence of hexanoate

enhanced

fatty

This indicates

from the mitochondria

637

of the lactate/

This corresponds

averaged 33 in the effluent

has been found during

recirculating reducing

(2.5 mM) led to elevation

that

the

medium. A similar acid oxidation

in

the transfer

of

to the cytoplasm must be

of

Vol. 41, No. 3,197O

rapid

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enough to maintain

system also.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a normal redox equilibrium

The subsequent addition

lactate/pyruvate

ratio

of ethanol

increased

the

AOOITION OF 1 mM lLC -CAPRONATE

f-l-5.2

U -6.7

-

further

perfusion

to about 80.

I

0.5

in this

W (--=

W

/--- -I : ; I II -56 I I

#---- II II! i .

5W-

“Co2

\ ---a

I ,

i-62%

W

I i -,,,, PVRUVATE

1

1I 36

1 5 I I1 II

COMPOSITION OF MEDIUM ENTERING THE

LIVER

ETHANOL WRUVATE LACTATE

(2.5mH) ( mM ) (mf4 )1

15

20

25

II I :

: : :

:, I8 I

-

-

+

0.1 1.0

0.1 10

Cl.1 1.0

30 I 1 I II I +

0.3 3.0

0.3 3.0

I

46 mln I 8

I 1I I I I ,

I +

1.0 10

¶.O 10

Fig. 2. Oxygen consumption, production of CO from capronate and levels of pyruvate as influenced by ethanol f n liters perfused once through with medium supplemented with three different levels of lactate and pyruvate. Lactate and pyruvate were added in the same proportion in all instances 40 that the lactate/pprurate ratio of the medium entering the liver was kept at IO. The levels of pyruvate are those of the medium leaving the liver.. The presentation is alightly schematic for the sake of clarity. It ie baeed on the mean values of three perfusions with identiaal patterns of directional changes. The numbers indicate the suppression induced by ethanol. 638

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Vol. 41, No. 3,197O

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The average oxygen consumption of livers and perfused thyroid rates

in

livers of oxygen

standard

conditions

showed increased

rates

and hypothyroid

oxygen consumption by almost 40 % (Fig. elevated

fasted

livers

regularly

(Fig.

slightly

investigations

2). When the pyruvate

level

2).

stimulating

indicate

effect

by ethanol

decreased stimulated

Addition

either

(Fig.

no influence

on hepatic

wa;

in both the

of ethanol

led to a small decrease in oxygen consumption

of the previous

Iiyper-

of 1 mMhexanoate

to 0.5 mMthe oxygen consumption also increased,

presence and absence of hexanoate

overnight

was 2.0 pmoles/g wet wt./min.

The addition

consumption.

from rats

(2.5 rd4)

2).

Most

or a

oxygen consumption

(1,2,3,10).

The production tivity

of 14C02 from ,-I4 C- acetate

(5 mM, specific

0.5 uCi/mmol) was suppressed 63 % by ethanol

tope dilution

due to ethanol-derived

acetate

(Table

is negligible

I).

acThe iso-

under these

Tab e 1. Suppression by ethanol of production of carbon dio ide from q-1 t C-acetate (5 mI6; specific activity 0.5 nCi/umol) and l- lG-hexanl,ate activity 5 nCi/umol) during nonrecirculating p$rfusion (I mM; specific of livers from hyper-, hypo-, and euthyroid rats. Mean values - S.D. of 3-5 perfusions are given.

Liberation

of 14c02

(cpm/g liver/min.) Substrate

Suppression by ethanol

Acetate

Presence of ethanol

+

Hexanoate

%

Suppression by ethanol

+

%

Type of liver Hyperthyroid Euthyroid Hypothyroid

84241 101t25

7120

72225

- 14

228+8g

I 45273

- 35

3794

- 63

195233

89:s

- 54

1323

- 81

224253

86224

- 62

639

Vol. 41, No. 3,197O

conditions.

BIOCHEMICAL

The corresponding

of l-14C-hexanoate (Table

mM, specific

(I

from hyperthyroid

of the oxidation hypothyroid

duction

livers

stronger

findings

of both extra-

in hyperthyroid afford

further through

higher

of a relation

acid

cycle

oxaloacetate,

these results

It

their

ratio

different

constant

(Fig.

of the oxidation

rates

level

level

of acetaldehyde

on the tricarboxy!atc

since simultaneously

and leaving,

it

employed in this

2).

of pyruvate

increased

the rate

(unpublished

of the pyruvate

was calculated

at the rate of ethanol

results).

disappearance

(I

It

the acti-

is stimu-

of metabolites

that

elimination

is therefore

in

at the highest of ethanol

umol/g/min.

is due to interaction

640

the

by providing

mM) addition of 1.8

Also,

to the

of capronate

in the level6

experiment

is ob-

in the pyruvate

reflect6

cycle

the oxidation

From the differences

leads to a reduction

relation

At

and at the

of hexanoate

can be seen (Fig.

this

levels 2).

redox change is diminished

is suggested that

of pyruvate

entering

and the

between the redox

between the decreases cause& by ethanol

by pyruvate.

pyruvate

(2,4,5),

in oxygen consumption seem to bear a relation

effect

pyruvate

rats

was diminished

between the redox state

but keeping

the ethanol-induced

levels.

the fluid

redox pairs

was made hy introducing

and in the 14 CO2 production

vating

towards re-

approach to the study of the relation

A relation

pyruvate

shift

In view

acid cycle.

and pyruvate

fluctuation6

(Table 1).

the ethanol-induced

same time a weaker suppression

levels

induced suppression

and hexanoate was much weaker and in

and intramitochondrial

the citric

levels,

served.

the ethanol-

than in normal livers

that

evidence

and the citric

of lactate

part

of the oxidation

5 ~Ci/mmol) was 54 %

but enhanced in hypothyroid

A different

lated

by ethanol

activity

rats

of both acetate

of the previous

state

suppression

1).

In livers

flux

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The high

and the output anticipated

that

between the

BIOCHEMICAL

Vol. 41, No. 3, 1970

alcohol

dehydrogenase and lactate

formed during pyruvate

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

the oxidation

reduced

of

dohydrogenase systems,

ethanol

being

rapidly

the NADH 2

reoxidized

when

to lactate.

REFERENCES

1. 2. 3. 4.

2: 7. 8.

Forsander, O.A., Riiihii, N., Salaspuro, M., and MYenpaI, P., Bi0chem.J. 22, 259 (19651. Ylikahri, RX., Maenpiiii, P.H., and Hassinen, I.E., Ann.Med.ExP. Biol.Fenn. M, 137 (1968). Williamson,-J.R., Scholz. R., Browning, E.T.. Thurman. R.G.. and Fukami, M.H., J.Biol.Chem. 244, 5044 Ti9691.' Lindroe, K.O., Eur.J.Bioche6?=1>, 111 (1970). Rawat, A.K. and Lundqvist, F.,=Eur.J.Biochem. 5, 13 (1968). Exton, J.H. and Park, C.R., J.Biol.Chem. 242, 2622 (1967). Scholz. R. and Biicher. Th.. in Control of-Eiiernv Metabolism. ed. bg'B. Chance, R.W; Es&brook and J.R. Williamson. Academic Press. New York 1965 D. 393. Hohorst, H.J., Kreutzi F:H:, and Biicher, Th., Biochem.2. 332 -e-v --18 (1959).

9. 10.

Williamson, J.R., Kreisberg, R.A., and Felts, P.W., Proc.Nat. Acad.Sci. 26, 247 (1966). Scholz, R.;- in Stoffwechsel der isoliert perfundierten Leber. hrsgg. W. von Staib und R. Scholz. Springer Verlag, Berlin 1468 P. 25.

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