Progress in Hepatic Encephalopathy and Metabolic Nitrogen Exchange

by Bengt Jeppsson
published: April 24th 1991 by CRC
binding: Hardcover, 592 pages
isbn: 0849301807 (isbn13: 9780849301803)

Chapter VII. Therapy in H.E.

POSSIBLE PROTECTIVE EFFECT OF L-CARNITINE ON HYPERAMMONEMIC

ENCEPHALOPATHY IN RATS

Ivo van der Lee, Joost Oudejans, Diederik K. Bosman, Jan G. de Haan, George G.A. Jörning,
Robert A.F.M. Chamuleau.

J. van GooI Laboratory Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 
1105 AZ Amsterdam, The Netherlands.
 313

POSSIBLE PROTECTIVE EFFECT OF L-CARNITINE ON

HYPERAMMONEMIC ENCEPHALOPATHY IN RATS

Ivo van der Lee, Joost Oudejans, Diederik K. Bosman, Jan G. de

Haan, George G.A. Jörning, Robert A.F.M. Chamuleau.

J. van GooI Llboratory

Academic Medical Centre, University of Amsterdam, Meibergdreef
9, 1105 AZ Amsterdam, The Netherlands.

I. INTRODUCTION

Portal systemic encephalopathy (PSE) is a neuropsychiatric syndro­

me which develops when the liver function has become so bad that
normal functioning of the brain cannot be maintained. Ammonia is
considered to play a key role in the pathogenesis of PSEY
Possible neurotoxic effe cts of hyperammonemia are inhibition of
ATP-synthesis, inhibition of cerebral blood flow, disturbance of
neurotransmitter baJance and inhibition of neuronal chloride trans­
port. 4 The exact mechanism of ammonia-neurotoxicity in PSE is still
a matter of debate. Whatever the mechanism may be, therapeutic
measures which lower plasma ammonia turned out to be important
in the treatment of hepatic encephalopathy.
The work of O'Connor et al has shown that administration of L-car­
nitine is able to protect mice against acute ammonia intoxicationY
They found that injection of L-carnitine (16 mMol/kg bw i.p.) 30
minutes before a LD100 of ammonium acetate (12 mMol/kg bw)
resulted in a 100% survival. Furthermore, suppletion of L-carnitine
10 minutes af ter a single dose of ammonium acetate resulted in a
50% reduction of the brain ammonia concentration. The mechanism
by which L-carnitine protects against the neurotoxicity of hyperam­
monemia is unknown . Several hypotheses have been proposed:
restoration of the possibly by hyperammonemia impaired malate­
aspartate shuttle,' and/or correction of the mitochondrial NADH/N­
AD- ratio by increased oxidation of long-chain fatty acids in the
presence of increased carnitine levels,! or neuro-excitatory effe cts of
L-acetyl carnitine on cholinergic cortical neurons based on structural
resemblance of L-acetyl carnitine and acetylchoJine .6
Whatever the explanation of the protective effect may be, the
promising resuJts of O'Connor et al prompted us to investigate the
protective effect of L-carnitine on acute and subacute hyperammo­
314 Progress in Hepatic Encephalopathy and Metabolic Nitrogen Exchange

nemic encephalopathy in the rat. In order to quantify such an effect

we have used our formerly described parameters of HE: EEG
spectral analysis, clinical grading and biochemical blood analysis. 7

IJ. MATERlALS AND METHOOS

A. ANIMALS
Male Wistar rats (200-275 gr, HSO Zeist, The Netherlands, 12 hr
light cycle: 8 a.m.-8 p.m.) were used in all experiments. The animals
we re fed standard laboratory chow (RM 1410, Hope Pharms, The
Netherlands) and water ad libitum. Animal care was taken according
to the animal welfare guidelines of the University of Amsterdam.

B. EEG-ANAL YSIS
At least one week before the start of the experiments four skull
electrodes were implanted and EEG analysis was performed. The
EEG signal was amplified (Nihon Kohden) and passed through a
Butterworth filter (Iow pass, cut off frequency 50 Hz with a roll off
of 48 dB/ octave) and analyzed on line. EEG slow wave activity was
defined as the ratio between low frequency power (1.0-7.4 Hz) and
high frequency power (13.5-26.4 Hz), the so called EEG left index. 8
This EEG left index was used to quantify the EEG abnormalities.

C. CLINICAL GRADE
The clinical grading of the severity of HE was defined as given in
Table 1.

TABLE 1

Stages in hepatic encephalopathy

grade 0: normal behaviour

grade 1: mild lethargy
grade 2: decreased motor activity, poor posture control
grade 3: severe ataxia, no spontaneous righting reflex
grade 4: no righting reflex on painful stimuli
grade 5: coma, no reaction on painful stimuli

D. STATlSTICS
Data are given as mean + / - SEM. Significance analysis has been
done by Student's t test. 9 p-Values below 0.05 are considered to be
significant.
 315

E. FIRST SERIES (acute hyperammonemia)

Sixteen hours before T=O all rats were injected i.p. with urease

(1000 U/kg bw). Two hours before the start of the experiment three

cannulas were inserted into respectively the carotic artery, the

jugular vein and the peritoneal cavity. Thirty minutes before the

start of t.he experiment the rats were injected with L-carnitine (8 or

16 mMol/kg bw i.p.) or an equivalent amount of saline. On T=O the

rats were injected with ammonium acetate (7 mMol/kg bw). Blood

(0.5 mi) was sampled from the carotic cannula for measuring the

plasma ammonia concentration at different time intervals as indica­

ted.

F. SECOND SERIES (subacute hyperammonemia)

Two or three days before the beginning of the experiments portaca­

val shunts were performed. 1o

On the day of the experiment an initial EEG was made before three

cannulas were inserted under ether anesthesia as indicated in series

1. The rats were allowed to recover for about 1Y2 hrs.

In order to obtain a steady state of hyperammonemia a loading dose

of ammonium acetate (004 mMol/kg bw) was given i.v. and a conti­

nuing dose of ammonium acetate of 1.9 mMollkg bw per hour. Twice

an hour an EEG-analysis was made and the clinical grade (Tabie 1)

was defined. Arterial plasma ammonia concentration was followed

regularly by the enzymatic Monotest of Boehringer Mannheim in iee

chilled plasma.

Four hours after the start of the experiment, when a steady state of

plasma ammonia was obtained, L-carnitine (16 mMol/kg bw) was

administered i.p. or an equivalent volume of NaCI 0.9% (controls).

EEG-Analysis and clinical grading was done every 10 min thereafter.

111. RESULTS

A. FIRST SERIES

The plasma ammonia concentrations of the saline and carnltIne

treated rats are shown in Figure 1. Only in the presence of 16 mMol­

/kg L-carnitine a significant decrease in plasma ammonia is obser­

ved at 10 and 20 min. ("." means p < 0.05)

The EEG left index both in the absence and presence of L-carnitine

is shown in Figure 2. The EEG left index is presented as percentage

of normal values.

316 Progress in Hepatic Encephalopathy and Metabolic Nitrogen Exchange

4000

:J
..... c a'n8 (n=4)
3200
Ö
3 sal ine (n=7)
2400

~ 1600

~<{
800
~

0
0 30 60 90 120
TI~ (m,n)

Figure 1: Acute hyperammonemia. Carni tine effects on

plasma ammonia.

400

320

0~

~ 240

r- 1\
LI. 1 \
w
...J
0

160

~
1'"
'>[.-"'1
....\ /
//[------1--­
I
W
w * ~} r/
~ ... ::,r--x ___ L __ l_
80

.L \* *car 16 (n=5) *
-
0

0 30 60 90 120

Figure 2: Acute hyperammonemia. Carnitine effects on EEG

left index.

L-earnitine as su eh has no influenee on the EEG left index (not

shown). A dose of 16 mMol/kg bw L-earnitine results in a normali­
zation of the left index in the period 30-120 min af ter ammonium
 317

acetate administration, whereas 8 mMol/kg L-carnitine showed

values in between saline and L-carnitine (16 mMol/kg) treatment.
Unfortunately th is EEG improvement is not reflected in the clinical
grading (Figure 3) .

The development of the clinical grade of HE was even more rapid in

the L-carnitine 16 mMol/kg group in the first 45 min. Thereafter
clinical HE grade was similar in all groups. Within 5 to 20 minutes
the rats became drowsy and reached grade 3 of HE for another 90
min.

5 ,-----------------------------------~
T I / saline (n=7)
__ l_ .. J
4 J
I \

~
g
3
,/ / -'1-- j!'t:::.L.,J__ -l___
~ f-- --­
2

,
T'
I' carn 16 (n=5l 1
(n=4)
,
I

I
04--L~ __ ~ ____ ~ ___ L_ _ ~ _ _ _ _~_ _ ~_ _~

o 30 60 90 120

TIIvE (min)

Figure 3: Acute hyperammonemia. Carni tine effects on

clinical grading.

B. SECOND SERIES
Plasma ammonia concentration reaches a steady state concentration
of 400-600 ,uM after 120 min. No significan~ difference between the
two groups is observed (Figure 4) .
During 240 min of hyperammonemia the left index (Figure 5) and
the clinical grade (Figure 6) of all rats show an increase. Fifteen min
af ter administration of L-carnitin~ (at T = 240, arrow) the left index
and the clinical grade seem to reach a plateau phase, but no signifi­
cant differences between the two groups are observed.
318 Progress in Hepatic Encephalopathy and Metabolic Nitrogen Exchange

1000

I
:)
~
0
2
.2
800

""
I
........
....
/

carn16 (n=4)

600

~
~~~

<l: 400 /
\ saline (n=4)
/
<l: /
2
U)
/

<l:
200
tr
0
0 80 160 240 320 400

TINE (mln)

Figure 4: Subacute hyperammonemia. Carnitine effects on

plasma ammonia.

700

560
l saline (n=4)
x
2
I­
420

l.L
III 280
..J
~
III
III
140

0
0 80 160 240 320 400

TINE (min)

Figure 5: Subacute hyperammonemia. carnitine effects on

EEG left index.
 319

5r-------------------------------------~

4
saline (n==4)

cam 16 (n=4)

OL-------~----~L_ ____ ~ _______ J_ _ _ _ _ __ J

o 80 160 240 320 400

TltvE (min)

Figure 6: Subacute hyperammonemia. Carnitine effects on

clinical grading.

IV. DISCUSSION

The striking protective effect of L-carnitine against the tOXIClty of

hyperammonemia in mice as shown by O'Connor et al,I.2 has not been
confirmed in our experiments in rats. Only a protective effect on the
EEG slow wave activity could be observed in acute hyperammo­
nemia: the increase in EEG left index af ter ammoniumacetate
administration was significantly shorter of duration in the treated
group. In contrast to saline treated animals the EEG left shift
normalised within 30-45 min, whereas peak levels of plasma ammo­
nia were significantly less in the first twenty minutes in the presence
of L-carnitine (16 mMol/kg). A lower dose of L-carnitine
(8 mMol/kg) induced EEG left index changes in between saline­
treated and high-dose carnitine rats.
Unfortunately this improvement of the EEG by L-carnitine during
acute hyperammonemia was not reflected in the clinical HE-grade:
up to 2 hrs af ter acute ammonium acetate loading the animals were
still in coma grade 3. In addition no significant effect of L-carnitine
320 Progress in Hepatic Encephalopafhy and Mefabolic Nifrogen Exchange

(16 mMol/kg ow) was ooserved on suoacute hyperammonemia in

portacaval shunted rats; neither EEG left index nor clinical grading
changed significantly during L-carnitine administration. Furthermore
in these experiments L-carnitine had no effect on the plasma ammo­
nia concentration.
Our data are in good agreement with those of Hearn et al,12 who also
showed a variabie protective effect of L-carnitine (16 mMol/kg ow)
administered 1 hr prior to a LDIOO of ammonium acetate in rats.
This variaole protective effect of L-carnitine was also short-lived
and completely lost if ammonium acetate was given 24 hrs af ter
L-carnitine administration.
The question still remains how to explain_ such a partial protective
effect of L-carnitine on hyperammonemic-induced encephalopathy in
the rat. The answer remains open as long as the mechanism of the
neurotoxicity of ammonia is still a matter of debate. Possible expla­
nations are interferences of L-carnitine with the by ammonia indu­
ced decreased inhibitory post synaptical potential, or restoration of
the increased cytosolic redox state resulting in increased brain
lactate concentrations during hyperammonemia as has been obser­
ved by many others.J·'·II.IJ
Although L-carnitine has a small peripheral effect in acute hype­
rammonemia in rat (series 1) as shown by a small but significant
decrease in plasma ammonia concentration in the first 20 min, it is
very unlikely that this observation could explain the central effect:
EEG left index normalized in the L-carnitine 16 mMol/kg group
whereas plasma ammonia (and therefore brain ammonia) was still in
a neurotoxic range (more than 1000,uM).'
Finally, it remains unexplained why the protective effect of L-carniti­
ne is much more pronounced in mice than in rats. More studies are
needed to answer all these questions.

V.REFERENCES

1. O'Connor, J.E., Costell, M., Grisolia, S., Prevention of ammonia

toxicity by L-carnitine: metabolic changes in brain, Neurochemical
Research, 9, 563, 1984.
2. O'Connor, J .E., Costell, M., Grisolia, S., Protective effect of
L-carnitine on hyperammonemia, FEBS letters, 2, 331, 1984.
3. Conn, H.O., Lieberthal, M.M., The hepatic coma syndromes and
lactulose, W illiam and W ilkins, Baltimore, 1979.
4. Cooper, A.J.L., Plum, F., Biochemistry and physiology of brain
ammonia, Physiological Reviews, 67, 440, 1987.
 321

5. Costell, M., Miguer, M.P., O'Connor, J.E., Grisolia, S., Effect of

hyperammonemia on the levels of carnitine in mice, Neurology, 37,
804, 1987.
6. Onorfry, M., Effect of L-acetyl carnitine HCI on rat steady-state
visual evoked potentiais. Comparison with L-carnitine, Drugs exptl.
din. res., 1, 5, 1987.
7. Chamuleau, R.A.F.M., Deutz, N.E.P., De Haan, J .G., Van Gooi,
J., Correlation between electroencephalographical and biochemical
indices in acute hepatic encephalopathy in rats, J. of Hepatology, 4,
299,1987.
8. Kropveld, D., Chamuleau, R.A.F.M., Popken, R.J., Smit, J., A
computerised EEG analysis system for smal\ laboratory animais,
Neuropsychobiology, 9, 159, 1983.
9. BMDP Statistical software package, Manual: Dixon, W.J., Uni­
versity of California press, Berkeley Los Angeles, 1984.
10. Hess, F., Shunts in the portal area of the rat,in Handbook of
microsurgery, 2, Olszewski, W.L., CRC Press, Florida, 301, 1984.
11. Deutz, N.E.P., Pathophysiological aspects of acute hepatic
encephalopathy in the rat, thesis University of Amsterdam, Studio
Intervisie, Ermelo, 1988.
12. Hearn, T.J., Coleman, A.E., Lai, J.e.K., Griffith, O.W., Cooper,
A.J.L., Effect of orally administered L-carnitine on blood ammonia
and L-carnitine concentrations in portal-caval shunted rats, Hepato­
~, 10, 822, 1989.
13. Deutz, N.E.P., Chamuleau, R.A.F.M., De Graaf, A.A., Bovée,
W.M.M.J., De Beer, R., In vivo lip NMR spectroscopy of the rat
cerebral cortex during acute hepatic encephalopathy, NMR in
Biomedicine, 1, 101, 1988.