Protection against acute hyperammonemia: The role of quaternary amines

Toxicology, 49 (1988) 83--90 Elsevier Scientific Publishers Ireland Ltd.

PROTECTION AGAINST ACUTE HYPERAMMONEMIA: THE ROLE OF QUATERNARY A M I N E S

OTMAR KLOIBER", BORISLAV BANJAC b and LESTER R. DREWES b

°Maz-Planck-Institute for Neurological Research, Cologne (F.R.G.) and bDepartment of Biochemistry and Chemical Toxicology Research Center, University of Minnesota, Duluth (U.S.A.)

SUMMARY

The quaternary amine L-carnitine is able to protect Swiss Albino mice from hyperammonemia when administered in high doses before ammonium acetate. This has been explained by its specific ability to shuttle fatty acids into mitochondria. The structure of L-carnitine resembles the chemical structure of other substances that have been described as being able to protect living cells against osmotic stress. We subjected Swiss Albino mice to hyperammonemia after pretreatment with L-carnitine or "osmoprotectants" such as the quaternary amines choline and betaine, and trimethylamine N-oxide. L-Carnitine proved to be the drug of choice to protect against acute hyperammonemia. Nevertheless, the other tested compounds appeared also to be effective, suggesting that osmoregulation plays a major role in protection against hyperammonemia.

Key words: Hyperammonemia; Osmoregulation; Choline; Trimethylamine N-oxide

L-Carnitine;

Betaine;

INTRODUCTION

Concepts concerning the mechanisms of protection against hyperammonemia have existed for many years. They are focused mainly on the urea cycle, and the theories suggest that protection occurs by increasing the ornithine availability thereby facilitating the turnover of ammonia groups via the urea cycle (see Ref. 1 for review). It is well known that Lcarnitine, a naturally occurring, nearly ubiquitous compound, shuttles the inward transport of activated long-chain fatty acids across the inner *Address all correspondence and reprint requests to: O. Kloiber, Max-Planck-Institute for Neurological Research, Ostmerheimer Str. 200, 5000 K61n 91, F.R.G. 0300-483X/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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mitochondrial membrane (see Ref. 2 for review). Furthermore, carnitine seems to enhance oxidative lipid metabolism [3], and it can modulate the cytoplasmic concentration of acetyl-CoA [4]. Recently O'Connor and coworkers [5] described the very successful t r e a t m e n t of acute ammonia intoxication with L-carnitine. The injection i.p. of 16 mmol/kg L-carnitine, given to Swiss Albino mice 30 min prior to administration of a lethal dose of 12 mmol/kg ammonium acetate, was able to prevent neurological symptoms, such as seizure or coma, and mortality. Costell et al. [6] found a higher production rate for urea but no stimulation of urea synthesis via N-acetylglutamate with carnitine and ammonia. Although the brain ammonia concentration (approximately 2 pmol/g) was reduced to about half that in unprotected mice, this reduction would not be expected to eliminate all neurological symptoms. The same group of authors proposed that L-carnitine induces the generation of reducing equivalents in mitochondria to mitigate the ammonia-induced blocking of the "malate-aspartate shuttle" and leads to an increased ATP production by oxidative phosphorylation [7]. Another explanation for the pronounced effects of L-carnitine was presented by Bobyleva-Guarriero et al. [8]. They speculated that the site of carnitine protection against hyperammonemia is not the brain but the liver. While studying isolated liver mitochondria after ammonia intoxication, they observed an improvement in depressed oxidative functions resulting from Lcarnitine. This was in accordance with the facilitated removal of NH4 ÷ from the organism as also reported by O'Connor et al. [5,7]. The molecular structure of L-carnitine resembles in many respects the molecular structures of the so-called "osymolytes" or "osmoprotectants" (Fig. 1). Most of them are quaternary amine compounds characterized by a terminal carboxy- or hydroxy group in contrast to other quaternary amines without oxygen or with an ester group (highly active compounds, e.g. choline esters). In this study we show that 3 (in microorganisms) established osmoprotectants are also protective against ammonia intoxication. Although these drugs have some toxicity themselves, they were highly protective against hyperammonemia. It is suggested, therefore, that L-carnitine and similar drugs act as osmoprotectants during acute hyperammonemia.

CH3

I

CH3 - N+ - CH3

I

CH2

CH3

CH3

CH3

I

I

I

CH3- N+-CH3

CH3-N +-CH3

CH3- N+-CH3

II

O

I

CH2

I

I

CH2OH

CHOH

I

CH2

I

COOH

I

CH2

I

COOH L-Carnitine

TMAO

Choline

Betaine

Fig. 1. Chemical structures of the tested compounds. Note the trimethylated ammonia and the terminal oxygens. (TMAO, trimethylamine N-oxide).

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MATERIALS AND METHODS

Male adult Swiss Albino mice were used for all experiments. In preliminary experiments, toxicity ranges were selected from available data for each test compound [9,10] to determine the maximum injectable doses. L-Carnitine was a generous gift from Professor Frederick E. Samson, University of Kansas. All other chemicals were purchased from Sigma Chemicals, St. Louis, MO. Animals were randomly subjected to one of the following treatments:

Control group (no pretreatment) Mice were injected i.p. with 12 mmol/kg ammonium acetate (0.8 M solution). Physiological Saline Animals were pretreated i.p. with 0.3 ml of saline solution. Thirty minutes later they were injected i.p. with 12 mmol/kg ammonium acetate (0.8 M solution). Betaine {glycine betaine) Animals were pretreated i.p. with 16 mmol/kg betaine hydrochloride (200 mg/ml) (brought to pH 3.0 with NaOH). Thirty minutes later they were injected i.p. with 12 mmol/kg ammonium acetate (0.8 M solution). Choline Animals were pretreated i.p. with 1.6 mmol/kg choline chloride (66 mg/ml) (brought to pH 5.8 with NaOH). Fifteen minutes later they were injected i.p. with 12 mmol/kg ammonium acetate (0.8 M solution). Trimethylamine N-oxide fTMA O) Animals were pretreated i.p. with 8 mmol/kg TMAO (100 mg/ml). Thirty minutes later they were injected i.p. with 12 mmol/kg ammonium acetate (0.8 M solution). L-Carnitine Animals were pretreated i.p. with 16 mmol/kg b-carnitine (200 mg/ml). Thirty minutes later they were injected i.p. with 12 mmol/kg ammonium acetate (0.8 M solution). In a first series of experiments, animals were observed for the onset of seizures, coma, and death. Animals surviving ammonium acetate treatment for 30 rain were killed under halothane anesthesia. Treatment groups with 10 consecutive surviving or non-surviving animals were terminated (control group, TMAO, and L-carnitine group). Other groups were continued to n = 15. In a second series of experiments, all animals in a treatment group (n -

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10) were frozen in liquid nitrogen 7 min after the i.p. injection of ammonium acetate. Prior to freezing, all animals were anesthetized for a maximum period of 1 rain by placing them in a chamber containing 40/0 halothane. Brains were removed in a freezer at - 2 0 ° C and powdered in a liquid nitrogen-cooled mortar. Aliquots of each powdered brain were weighed and deproteinized with acetonitrile (1 ml/75 mg tissue) for the analysis of amino acids. Amino acids were determined using HPLC as previously described [11]. RESULTS

The compounds tested were able to protect against hyperammonemia induced by the i.p. injection of 12 mmol/kg ammonium acetate. As described by O'Connor and co-workers [5] all mice receiving 16 mmol/kg L-carnitine survived, as did the animals treated with 8 mmol/kg TMAO. In the choline group, 2 out of 15 animals died, and in the betaine group, 5 out of 15 died. Surprisingly, we found that not all the animals receiving only saline solution before ammonium acetate died (5 out of 15 survived). However, there were no surviving animals in the untreated group, which received only ammonium acetate (Fig. 2). In the untreated group, the hyperammonemia resulted in rapidly onsetting coma after a brief hyperexcitable period followed by short clonic seizures that could be triggered by noise. Death occurred between 7 and 13 min. Of the 15 animals in the saline group, 10 animals died, 9 had seizures, and one was comatose at the end of the experiment. In the betaine group,

Protection Against Hyperammonemia =n

100 80

.o G) a.

60 • []

40

died survived

20 0 Control

Saline

Betaine

Choline

T M A O Carnitine

Fig. 2. Protection against hyperammonemia by quaternary amines. Swiss Albino mice were injected i.p. with 12 mmol/kg ammonium acetate 30 min after receiving saline, betaine, trimethylamine N-oxide (TMAO), or L-carnitine or 15 rain after receiving choline. The control group received only ammonium acetate. Note the 100% survival rate with TMAO and Lcarnitine.

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TABLE I GLUTAMATE/GLUTAMINERATIOS (Means ± S.D., ~mol/g wet wt)

Control Saline Betaine Choline TMAO L-Carnitine

Glutamate

Glutamine

Glu/glu-N

12.26 12.52 15.00 12.33 16.17 14.59

5.88 6.36 6.70 6.22 7.09 7.33

2.1 2.0 2.2 2.0 2.3 2.0

~- 0.56 ± 0.42 ± 0.41 ± 0.37 ± 0.71 ± 0.64

± ± ± ± ± ±

0.13 0.22 0.27 0.21 0.50 0.29

none of the 10 s u r v i v o r s had seizures, b u t 2 a p p e a r e d to be h y p e r e x c i t a b l e , and one of those was c o m a t o s e at 30 rain. Out of the 5 t h a t did not survive, 2 had seizures. In the choline group, 8 of the 15 animals w e r e hyperexcitable, and 2 of t h e m died. In t h e g r o u p t r e a t e d with TMAO, 5 of the 10 animals w e r e h y p e r e x c i t a b l e , and 2 had seizures. One of the h y p e r e x c i t a b l e and one of the seizuring animals w e r e c o m a t o s e at the end of the o b s e r v a t i o n period. In the L-carnitine-treated group, none of t h e animals e x p e r i e n c e d seizures, and only 2 a p p e a r e d to be h y p e r e x c i t a b l e ; otherwise, the animals b e h a v e d normally. Amino acid levels in brain tissue generally increased a f t e r t r e a t m e n t with q u a t e r n a r y amines or TMAO. The ratios of g l u t a m a t e / g l u t a m i n e and a s p a r t a t e / a s p a r a g i n e w e r e not a l t e r e d by any t r e a t m e n t (Tables I and II). DISCUSSION L-Carnitine's p r o t e c t i o n a g a i n s t h y p e r a m m o n e m i a has been discussed from different viewpoints since its discovery. It has been s u g g e s t e d t h a t elevated

TABLE II ASPARTATE/ASPARAGINE RATIOS (Means ± S.D., ~mol/g wet wt)

Control Saline Betaine Choline TMAO L-Carnitine

Aspartate

Asparagine

Asp/asp-N

2.13 2.14 3.11 2.42 3.54 2.45

0.074 0.076 0.092 0.096 0.088 0.090

28.8 28.2 33.8 25,2 40,2 27,2

± ± ± ± ± ±

0.13 0.07 0.08 0.05 0.13 0.10

3= 0,004 ± 0,004 ± 0,004 ± 0,004 ± 0.006 ± 0,003

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ammonia in brain tissue leads to a reduced A T P production by inhibiting the malate-aspartate shuttle of reducing equivalents from cytoplasm into mitochondria and by removal of intramitochondrial NADH via the glutamate dehydrogenase reaction [12]. O'Connor and co-workers [7] proposed that Lcarnitine could induce an intramitochondrial generation of reducing equivalents (NADH) which could overcome the ammonia-induced blocking of the malate-aspartate shuttle. They described an improved glutamate/ glutamine ratio with L-carnitine treatment, which we could not confirm. Although they found decreased tissue ammonia levels and increased urea production after treatment with L-carnitine, they could not find a stimulation of urea synthesis via N-acetyl-glutamate. According to studies performed with isolated liver mitochondria from animals subjected to hyperammonemia and L-carnitine treatment [8], Lcarnitine is able to preserve the permeability of mitochondrial membranes otherwise impaired by ammonium acetate. This might result from a removal of long-chain acyl-CoA from the mitochondrial membrane [13]. Microorganisms, plants, and animals have developed different strategies to survive water stress [14,15]. A common mechanism seems to involve the use of "osmolytes" or "osmoprotectants," which can enable the organism to withstand extreme osmotic conditions. In vertebrates, this strategy has been demonstrated for the balancing of high urea concentrations in kidneys [16]. Urea concentrations of 0.4 M and higher, normally incompatible with any enzyme function, are tolerated by cells of the kidney if trimethylamine compounds, e.g. TMAO, are present at a 2 to I ratio (ureafrMAO). Osmoprotective quaternary amines such as glycine betaine, betaine aldehyde, choline, butyrobetaine, proline betaine, and TMAO all have terminal oxygen, carboxy- or hydroxy groups. An exception seems to be glycerophosphorylcholine which has non-terminal oxygens. L-Carnitine contains structural similarities to these compounds and has both a terminal carboxy group and a hydroxy group in the fl position (Fig. 1). Betaine, choline, and TMAO were able to mimic L-carnitine function in ammonia protection. We suggest that the protective effect of these compounds against acute hyperammonemia is a result of their function as osmoprotectants. It may be that enzymes or other proteins are stablilized by their presence. The protective effect is unlikely a result of their ability to shuttle fatty acids across the mitochondrial membrane, which is a known function of L-carnitine. The fact that betaine and choline did not protect as completely as Lcarnitine or TMAO must be considered from 2 viewpoints. It might be possible that L-carnitine reaches the mitochondria more rapidly because of its specific shuttling function and, therefore, is more effective than any other compound. However, since we did not do toxicity/effectivity studies for tile other tested compounds, their concentrations most likely were not in the optimal therapeutic range, if one exists. Choline was effective at a concentration 10-fold lower than L-carnitine. This can be explained by an existing transport system for choline at the blood-brain barrier [17,18] and the extremely slow blood-brain transport of L-carnitine [19]. Osmoprotection

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as proposed here is certainly not a feature limited to the brain; however, the absence of any neurological symptoms with L-carnitine treatment or with TMAO, even under markedly elevated brain ammonia concentrations, points out stabilizing effects from these substances in the central nervous system. At this point the mechanism of protection by saline against hyperammonemia is unknown. Further studies may provide explanations for this effect as well as for the long-term result of the protection by the other described substances. It will also be of interest to see any similarities and differences between the protective effects by the quaternary amines and the well-known therapeutic strategies in dietary management of ammoniaendangered subjects. ACKNOWLEDGEMENTS

The authors wish to thank Carolyn Clark for editorial assistance and preparation of the manuscript, William R. Bailey for technical assistance, and the Duluth Clinic Foundation for its financial support. REFERENCES 1

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