Ammonia detoxification in the fatty liver

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 107, No. 4, 1982

Pages 1440-1445

August 31, 1982

AMMONIA

DETOXIFICATION

Jonathan

H. B l a n d i n g

IN THE FATTY L I V E R 1

and George

C. T r e m b l a y

D e p a r t m e n t of B i o c h e m i s t r y and Biophysics U n i v e r s i t y of Rhode Island Kingston, P~ode Island 02881 Received July 16, 1982

SUMMARY: Fatty livers were p r o d u c e d by feeding w e a n l i n g rats a diet made 1% in orotic acid. Liver slices from these animals exh i b i t e d a sharply reduced c a p a c i t y for the i n c o r p o r a t i o n of (14C)NaHCO 3 into urea. However, the a b i l i t y of the intact animal to survive a single c h a l l e n g e of a m m o n i u m acetate was not comp r o m i s e d by fatty m e t a m o r p h o s i s of the liver; if anything, animals with the fatty liver seemed p r o t e c t e d against ammonia toxicity. INTRODUCTION: sponse

Fatty m e t a m o r p h o s i s

to the i n g e s t i o n

of a w i d e

these may be of d i e t a r y biotics,

methotrexate,

solvents, among

diseased

states

The e x t e n t

(alcoholic

origin

to w h i c h

changes

commonly

associated

to isolate

the usual necrosis

fatty

liver

diabetes,

(some anti-

(organic

is also

and other

complications are absent,

with

ammonia

liver

of lipid

by the p o t e n t i a l the fatty

the i n f l u e n c e

by e x a m i n i n g

The

A fatty

the a c c u m u l a t i o n

is o b s c u r e d

acid.

(1-4).

substances;

medicinal

or e n v i r o n m e n t a l

re-

found

less c o m m o n

(3).

function

function

is a common

of h e p a t o t o x i c

beverages),

of m a l n u t r i t i o n ,

hepatic

attempted

variety

salicylates),

pesticides)

the symptoms

of the liver

induced

compromises

involvement

liver

(3).

by orotic

of hepatitis,

and the animals

in rats

Copyright © 1982 by Academic Press, Inc. AII rights o f reproduction in any,form reserved.

1440

on liver fed orotic

acid is unique

fibrosis, appear

in that

cirrhosis,

and

to be in good health

iThis work was s u p p o r t e d by a Public H e a l t h Research (AM26166) from the N a t i o n a l Institute of Arthritis, Digestive, and Kidney Diseases.

0006- 29IX/82/161440-06501.00/0

We have

of fat i n f i l t r a t i o n

detoxification

of other

Grant Diabetes,

Vol. 107, No. 4, 1982 (5).

Ammonia

dependent

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

detoxification

is a v i t a l h e p a t i c

upon e n e r g y - c o n s u m i n g

and c y t o p l a s m i c

compartments

reactions

of the l i v e r

function which

in b o t h

is

the m i t o c h o n d r i a l

cell.

.MATERIALS AND METHODS: W e a n l i n g male rats of the S p r a g u e - D a w l e y s t r a i n w e r e o b t a i n e d from the C h a r l e s R i v e r Colony, Boston. Upon arrival, the rats were d i v i d e d into 3 g r o u p s and p l a c e d on one of the f o l l o w i n g diets for 14 days: (i) P u r i n a Rat C h o w 5012, (ii) a s e m i - s y n t h e t i c d i e t c o n s i s t i n g of sucrose, 69.5%; v i t a m i n - f r e e casein, 20.0%; D L - m e t h i o n i n e , 0.3%; c h o l i n e b i t a r t r a t e , 0.2%; Lc y s t i n e 0.5%; A I N m i n e r a l m i x 3.5%; A I N v i t a m i n mix, 1.0%; fiber 5.0%, or (iii) the s e m i - s y n t h e t i c d i e t s u p p l e m e n t e d w i t h o r o t i c a c i d by s u b s t i t u t i n g 1 g o r o t i c acid for 1 g s u c r o s e per 100 g diet. The s e m i - s y n t h e t i c diets w e r e p r e p a r e d by I C N - N u t r i t i o n a l B i o c h e m i c a l s , C l e v e l a n d , Ohio. The a n i m a l s w e r e fed ad libitum. The i n c o r p o r a t i o n of b i c a r b o n a t e into u r e a was m e a s u r e d in liver slices (500 mg, w e t weight) i n c u b a t e d w i t h ( 1 4 C ) N a H C O 3 (30 mM, 100-300 ~Ci) in 20 ml of Krebs I m p r o v e d R i n g e r II S o l u t i o n at 37oc for 3 h, as d e s c r i b e d p r e v i o u s l y (6). At the e n d of the i n c u b a t i o n period, r a d i o l a b e l e d u r e a was i s o l a t e d f r o m the a c i d s o l u b l e f r a c t i o n of the r e a c t i o n m i x t u r e by c o c r y s t a l l i z a t i o n w i t h c a r r i e r (7). A m m o n i a t o x i c i t y was d e t e r m i n e d w i t h a single i n t r a D e r i t o n e a l i n j e c t i o n of a m m o n i u m acetate in w a t e r (0.5M), a d m i n i s t e r e d in the volume r e q u i r e d to p r o v i d e a dose of 8.2 m m o l e s per kg b o d y weight; this dose is the r e p o r t e d LD50 (8). The c o n c e n t r a t i o n of a n ~ o n i a in the s o l u t i o n to be i n j e c t e d was v e r i f i e d b y N e s s l e r i z a t i o n . In the w h o l e animal studies, liver fat w a s d e t e r m i n e d on p o o l e d a l i q u o t s of 100 mg liver f r o m e a c h a n i m a l in the d e s i g n a t e d group. For the i n c o r p o r a t i o n studies, fat c o n t e n t was d e t e r m i n e d for the liver u s e d to p r e p a r e the slices. The fat c o n t e n t was m e a s u r e d g r a v i m e t r i c a l l y , a f t e r e x t r a c t i o n of the lipids f r o m an h o m o g e n a t e a c c o r d i n g to the p r o c e d u r e of F o l c h et al. (9).

RESULTS AND DISCUSSION: slices w e r e

acid

diets,

and s e m i - s y n t h e t i c

slices

f r o m the a n i m a l s of u r e a g e n e s i s to a m m o n i a

lated

incorporation

about

the same e x t e n t

incorporation

and a b o u t

diets,

another

The

(3.3-3.9 4.2-5.4

slices

acid

times 1441

I).

to r e s u l t Ammonium

of

(14C)on the

liver

However,

im-

from a l t e r e d

chloride on all

and o r n i t h i n e

over

for a n i m a l s

from animals

from a n i m a l s

times)

on the P u r i n a

reduced with

(Table

not a p p e a r

slices

greater

from which

incorporation

but sharply

and o r n i t h i n e .

in liver

for a n i m a l s

liver

fed o r o t i c does

of the livers

5 times

I).

similar with

Purina

sensitivity

the same

(Legend to T a b l e

N a H C O 3 into urea was

pairment

fat c o n t e n t

p r e p a r e d was a b o u t

and s e m i - s y n t h e t i c fed o r o t i c

The

stimu3 diets

enhanced

the rate o b s e r v e d

the

with

to

Vol. 107, No. 4, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE I

INCORPORATION OF

(14C)NaHCO3 INTO UREA BY LIVER SLICES Diet Semi-Synthetic

Semi-Synthetic with 1% Orotic Acid

1.03 -+ 0.4

0.96 ± 0.2

0.24 ± 0.i

3.40 ~ 0.3

3.78±1.7

0.87±0.4

Additions None

Purina

(control)

NH4CI, 5 mM NH4CI, 5 mM + Ornithine, 5 mM

18.50 + 3.4

16.00 ± 3.9

3.83 ± 1.6

The fat contents of the livers used to prepare slices were 3.5 • 0.i, 4.4 ~ 0.6, and 22.5 ~ 3.8% (average e sd) for animals on the Purina (n = 3), Semi-Synthetic (n = 5), and 1% Orotic Acid (n = 5) diets, respectively. The data shown are averages ± sd in micromoles (14C)NaHCO 3 incorporated into urea per g tissue during 3 h incubation.

NH4CI

alone.

in s l i c e s of

of the

regardless

fatty

alter

fatty

liver.

The

findings

f u s i o n of tatively

liver

isolated

ciency

(i0).

morphosis intact

intact could

Thus

their

However,

reasonably

of the

observed

animals

proceeded

to t e s t

ad

libitum

liver.

Since

fed a d i e t

occasions,

for

suggest

ammonia

14 d a y s

following

no s u c h

with

weanling

1442

these

ammonia

toxicity are

in the

too

orotic

the

of a m m o n i a

than

complications with

severely

with

a challenge other

defi-

fatty meta-

the a b o v e h y p o t h e s i s

supplemented

prior

found a quanti-

that

animals

fatty

have

been

acid,

animals.

rats were

to r e c e i v i n g

not

of s u p e r -

from choline

to c o m p l i c a t i o n s

the h y p o t h e s i s

On 4 s e p a r a t e diets

mortality

does

of n o r m a l

from exogenous

choline-deficient to t e s t

tissue

an a r t i f a c t

resulting

and ours

Expression

by slices

and B e c k e n h a u e r

liver

ureagenesis

75%.

g fat-free

to b e

exacerbate

be a t t r i b u t e d

metamorphosis

per

appear

results

symptoms

increased

by about

of u r e a g e n e s i s

fatty

conditions,

in u r e a g e n e s i s

Barak

liver would

by o t h e r

with

since

impairment

of the

animal;

do n o t

perfused

animal.

stressed

above

reduced

of a c t i v i t y

disparity

slices

similar

in the

the

of the a s s a y

liver was

the d a t a on the b a s i s

appreciably and

Thus,

fed

1 of

the

a challenge

of

3

we

Vol. 107, No. 4, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE

II

F A T T Y I N F I L T R A T I O N OF THE L I V E R A N D THE A B I L I T Y TO W I T H S T A N D A C H A L L E N G E P u r i n a Rat Chow

OF A M M O N I A Semi-Synthetic w i t h 1% O r o t i c A c i d

Semi-Synthetic Body Weight

Expt Expt Expt Expt

1 2 3 4

Average

~ sd

139 e 15g 149 ~ 12 135 ± 9

130 124 iii 115

± 17g e 14 ± 9 ± 8

139 + 13

118 ± 14

ii0 ii0 102 I00

± 9g ± 13 ± 7 ± ii

105 ± i0

Liver Weight Expt 1 Expt 2 Expt 3 Expt 4 Average

8.6 ± 0.5g 7.8 ~ i.i 6.4 ~ i.i

± sd

10.9 9.7 7.5 8.2

7.4 + 1.3

± + ± +

Average

~ sd

10.2 11.2 9.6 8.7

8.9 + 1.9 Liver

Expt 1 Expt 2 Expt 3 Expt 4

1.5g 1.7 1.4 i.i

+ ± + ±

0.9g 2.0 1.4 1.6

9.8 ~ 1.8

Fat

4.2% 3.9 4.0

5.6% 4.5 6.3 6.3

24.3% 20.2 22.5 21.4

4.0 ~ 0.2

5.7 ± 0.9

22.1 ± 1.7

Mortality Expt 1 Expt 2 Expt 3 Expt 4

9/20 4/10 6/20

i0/i0 8/10 11/20 7/10

3/10 5/9 12/21 4/10

Sum

19/50

36/50

24/50

Overall Mortality

38%

72%

48%

Four d i f f e r e n t shipments of w e a n l i n g rats w e r e p l a c e d o n the d e s i g n a t e d d i e t s for 14 days and then c h a l l e n g e d w i t h a s i n g l e dose of 8.2 m m o l e s of a m m o n i u m acetate, ip. E a c h e x p e r i m e n t was c o n d u c t e d w i t h a single s h i p m e n t of a n i m a l s and the n u m b e r of a n i m a l s in any g r o u p is shown in the d e n o m i n a t o r of the m o r t a l i t y rate. B o d y w e i g h t and liver w e i g h t are given as a v e r a g e s ± sd for e a c h g r o u p after 14 days on the diet; the i n d i v i d u a l v a l u e s w e r e a l s o p o o l e d to c a l c u l a t e the a v e r a g e ± sd for all a n i m a l s on the same diet.

ammonia.

Weight

consistently but

lower

consistently

(Table acetate fed

gain

II). (8),

animals

than

higher

When

the

30-57%

semi-synthetic

weight than

challenged

mortality and

on

gain

on

weight with

ranged

from

in

groups

the

1443

the

gain

the

diet Purina

on

the

reported

30-45% of

was

in

diet,

orotic LD50

the

orotic

slightly

for

groups

acid-fed

and acid

but modestly diet

ammonium of

Purina-

animals,

Vol. 107, No. 4,1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

yielding overall mortality rates of 38% and 48%, respectively (Table II).

The groups fed the semi-synthetic diet lacking orotic

acid, which are the appropriate controls for the groups fed orotic acid, showed the widest range in mortality

(55-100%) with an over-

all mortality rate of 72% for the 50 animals tested. Thus, raising the fat content of the liver from 5% to over 20% by feeding orotic acid clearly does not compromise the ability of the animal to withstand a challenge of ammonia;

if anything,

appears to offer protection against ammonia toxicity.

it

It seems

prudent to caution that metabolic changes brought about by dietary orotic acid might somehow compensate for the impairment of ureagenesis observed with liver slices.

The possibility that dietary oro-

tic acid afforded protection by virtue of its precursor relationship to pyrimidine nucleotides was examined by testing the influence of uridine on mortality following a challenge of ammonia

(uridine is

more soluble and more rapidly converted to pyrimidine nucleotides than is orotic acid).

When groups of 15 male rats weighing I00-

125 g were injected with uridine

(5 mmoles,

ip/kg body weight)

either i0 min or 1 h prior to the injection of ammonium acetate at either the LD50 or the LD99.9 not shown).

(8), mortality was not affected

(data

Thus, the ability of animals on the orotic acid diet

to withstand a challenge of ammonia, in spite of a sharply reduced capacity of liver slices from these animals for ureagenesis,

re-

mains to be explained.

References i. 2. 3. 4. 5.

Lieber, C.S. and DeCarli, L.M. (1977) Metabolic Aspects of Alcoholism, pp. 31-79, MTP Press, Ltd., Lancaster. Freeman-Narrod, M., Narrod, S.A., and Yarbro, J.W. (1977) Med. Pediat. Oncol. 3, 9-14. Hoyumpa, A.M., Greene, H.L., Dunn, G.D., and Schenker, S. (1975) Am. J. Dig. Dis. 20, 1142-1170. Rees, K.R. (1964) Metabolism and Physiological Significance of Lipids, pp. 443-453, John Wiley and Sons, Ltd., London. Jatlow, P., Adams, W.R., and Handschumacher, R.E. (1965) Am. J. Pathol. 47, 125-145. 1444

Vol. 107, No. 4, 1982

6. 7. 8. 9. 10.

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

Tremblay, G.C., Crandall, D.E., Knott, C.E., and Alfant, M. (1977) Arch. Biochem. Biophys. 178, 264-277. Wendler, P.A. and Tremblay, G.C. (1980) Anal. Biochem. 108, 406-418. Greenstein, J.P., Winitz, M., Gullino, P., Birnbaum, S.M., and Otey, M.C. (1956) Arch. Biochem. Biophys. 64, 342-354. Folch, J., Lees, M., and Stanley, G.H.S. (1956) J. Biol. Chem. 226, 497-509. Barak, A.J. and Beckenhauer, H.C. (1966) Biochem. Pharmacol. 15, 1295-1302.

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