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Metabolism and kinetics of parenterally administered ornithine and α-ketoglutarate in healthy and burned animals
CLINICAL
NUTRITION , Longman
(19.W) 7: 105-l 11 I.rd 1988
Group IJK
Metabolism and Kinetics of Parenterally Administered Ornithine and a-Ketoglutarate in Healthy and Burned Animals M. Vaubourdolle*, and L. Cynober* *Laboratoire
de Biochimie
A. Jardelt, CNRS
C. Coudray-Lucas*,
UA 622 and tLaboratoire
de Physiologie,
92296 Chatenay Malabry Cedex, France (Reprint requeststo M.V.)
0. G. Ekindjian*, Universitk
J. Agneray*
Paris XI, Tour D4,5
Rue JB Clkment,
To improve our understanding of the metabolism of ornithine a-ketoglutarate (OKG), ABSTRACT used as an adjuvant of parenteral nutrition, we studied the plasma kinetics, localisation of target tissues and metabolism of a ketoglutarate (aKG) and ornithine (Orn) in healthy and burned animals. After parenteral administration, the kinetics of plasma disappearance of the two labelled compounds showed a biphasic decrease reflecting an open two-compartmental model of elimination, with the exception of Orn in burned rats. The appearance of metabolites in the plasma was rapid, particularly with regard to glutamate, proline and, following ornithine administration, glutamine. This witnessed the engagement of the substrates in multiple metabolic pathways. The study of tissular distribution by autoradiography demonstrated certain target tissues in common for aKG and Orn such as the liver, intestinal mucosa, salivary glandsj kidney and muscle. This is consistent with a possible synergic action of these two compounds. The identification of labelled amino-acids and aliphatic polyamines was also performed in the tissues. Two major observations were made, which could be of interest in further work concerning cellular mechanisms of OKG action. Firstly, after aKG administration, labelled Glu and aKG were detected simultaneously in muscle; this would render possible the local biosynthesis of the anticatabolic compound a ketoisocaproate. Secondly, the injection of 14C-ornithine was followed by the appearance in the intestinal mucosa and salivary glands of labelled aliphatic polyamines, which possess an anabolic effect.
INTRODUCTION Ornithine r-ketoglutarate (OKG) has been used in the
treatment of traumatised patients as an adjuvant in parenteral and enteral nutrition with anabolic and/oranticatabolic effects [14]. These effects include a rapid improvement in the nitrogen balance [ 1, 3-51, a reduction in hyperphenylalaninemia [2] and an improvement in plasma levels of visceral proteins [3]. However, many questions concerning this compound remain unresolved. Firstly, although there are numerous in vitro studies on isolated cells or organs concerning ornithine and rKG metabolism, to our knowledge little work has been don: on the in vivo metabolism of administered Om and/or rKG. The only existing study performed in humans involved the oral administration of Om, clKG and OKG to healthy subjects [6]. An increase in plasma glutamate levels after OKG, aKG, and Om loads was observed, whereas Pro and Arg increased only after the OKG load. However, plasma amino-acid variations is the net re-
sult of interorgan exchanges. It thus appeared desirable to perform animal experiments in order to determine the role of the various organs in the metabolism of Orn and aKG administered in vivo. Secondly, the mechanism of OKG action is unknown. Although it has been postulated [l-2] that OKG exerts its anabolic/anticatabolic action via a stimulation of insulin or growth hormone secretion in traumatised patients, this hypothesis remains debatable [7]. Other possible mechanisms might involve either the formation of polyamines from ornithine [8] or the production of branched-chain ketoacids in the transamination reaction involving a-ketoglutarate (aKG) [9], or further the production of glutamine from both ornithine and aKG [lo]. In effect, ornithine and aKG possess partially common metabolic pathways [ 1 l] and, when administered simultaneously, the repercussions of each compound on the metabolism of the other are unknown. Another interesting point remains obscure: OKG administered to surgical [I] and bum patients [2] leads to abnormally high plasma and muscle concentrations of omithine and proline, its metabolite, compared
105
106
METABOLISM AND KINETICS OF PARENTERALLY
ADMINISTERED ORNITHINE AND 1-KETOGLUTARATE
to values obtained in experiments on healthy subjects [ 12, 131. The reasons for this accumulation of ornithine remain unclear. In order to investigate the metabolism of OKG, before going on to study to its mechanism of action, we studied here the plasma disappearance, target tissues and metabolites of the two components of the OKG molecule in healthy and burned animals using the parenteral route of administration.
MATERIALS
AND
METHODS
Kinetics and metabolic studies Male Wistar rats weighing 200-300g were fed a commercial pelleted diet (A03 regimen, UAR) ad libitum. At the beginning of the experiments, half the animals were anaesthetised with diethylether and burned by immersing the dorsum in water at 80°C for 10 s, producing a full-thickness scald injury over 20% of the body surface area [ 141. A volume of 0.15 M NaCl, corresponding to 1076 of the body weight, was administered parenterally in order to prevent any eventual state of shock [14]. The burned rats showed no sign of discomfort and did not receive pain medication. Three days following the thermal injury, all the rats were starved for 3 h and anaesthetised with thiopental (25mg/kg). The animals were cannulated in the right external jugular vein for intravenous injection and a catheter was placed in the left carotid artery for blood sampling. Then at (to), 25uCi of a-[U-14Qketoglutarate (14C-KG, spec. act. 258 mCi/mmol, NEN, Albany, USA) or 25 uCi of [U-14C]-ornithine (14C-Om, spec, act, 266 mCi/mmol, NEN, Albany, USA) was administered parenterally (volume 0.5ml) to the healthy and burned rats. Certain animals received 14C- KG (or 14C-Orn), diluted in a solution of unlabelled c1KG (or Orn) at a concentration corresponding to 0.5g of ornithine cr-ketoglutarate per kg of body weight, a dose used in human therapeutics [2]. Venous blood (0.25 ml) was collected in heparinised tubes at 0.5, 1, 2, 5, 10, 15 and 30 min after parenteral injection; the radioactivity present in 0.1 ml of plasma was measured in 5ml aqualuma solvent with a liquid scintillation counter. In some cases (see below), 0.1 ml of plasma was stored at -20°C for qualitative analysis of radioactivity. At the linal time (30 min), the rats were killed by decapitation and the liver, kidneys, salivary glands, soleus muscle and intestinal mucosa were collected. After weighing, each piece of tissue was homogenised at 4°C in 0.3M perchloric acid containing 0.5 mM EDTA (5 ml/g of tissue). After centrifugation (12 000 g, 15 min) and measurement of the total volume, the supematant was removed and divided into several
aliquots for the determination of total radioactivity and the qualitative analysis of labelled amino-acids. For the latter, bidimensional thin-layer chromatography (TLC) of dansylated amino-acids was carried out as described by Biou et al [15] on micropolyamide plates (F 1700, 5 x 5cm, Schleicher and Schiill, Dassel, FRG). The fluorescent spots were cut out and dissolved in 5ml scintillation fluid for counting. Amino-acids were identified by calculating the position of the spot, with reference to dansyl-glycine. The initial method was applicable for the detection and identification of labelled aliphatic polyamines (putrescine, spermine, spermidine). The minimal detectable amount was in the picomole range.
Whole body autoradiography Male Swiss mice weighing 20-22g were used for the experiments. The nutrition and thermal injury were as described above. On the day of the experiment, 25 uCi of 14C-KG or 14C-Orn, with or without unlabelled KG or Orn respectively, was administered parenterally (see above). Five, 10 and 15min following peritoneal injection or 2 and 5 min after intravenous administration, the mice were killed by immersion in acetone at -70°C and stored at -20°C for 1 week. Sagittal sections of the animals, 20 urn thick, were prepared with a microtome and dried at -20°C. The sections were placed on X-O- mat film (Kodak) for 3-4 weeks as described by Cohen and Delassue [ 161 and then developed. There were three animals in each of the above series, i.e., presence or absence of burns, addition or not of unlabelled compounds, peritoneal or intravenous administration.
Statistics Data are given as mean & SD. Comparisons were made by the Mann Whitney U-test and differences with p values of less than 0.05 were considered significant. The statistical analysis of the kinetics of plasma radioactivity was carried out by the method of residuals [ 171 and linear regression.
RESULTS Fate ofparenterally-administered labelled u-ketoglutarate The decrease in plasma radioactivity as a function of time after jugular injection of 14C-KG in healthy rats (n=6) is presented in Figure 1A. The experimental curve can be superimposed on the graphic representation of the equation: y = Ae + Be. This may reflect the existence of an open two-compartmental model for the
CLINICAL
Table 1 rats
Analysis
of plasma
radioactivity
%-ctKetoglutarate “C-ornithine
tcpm
*cpm > 800;, of total.
Analysis
Tissue Kidney Liver Salivary glands Intestinal mucosa
Muscle
of labelled
content
3~Ketoglutarate + + + +
+
injection
of labelled
Labelled
Time after injection (min)
Administered compound
Table 2 rats
after intravenous
products
2 30
+ +*
_ _
2 30
_ _
+t
or Orn in male Wistar
recovered Unidentified molecules
Amino-acids
Ornithine
a Ketoglutarate
rKG
_
I
Glutamate Proline _
+
107
NUTRITION
_. 1
Glutamate Glutamine Proline Arginine
> 600/, of total.
of tissues 30min
after intravenous
Ornithine
Proline
administration
Glutamate
_
_
_
_
+
-
_
+
+
+ +
+
elimination of this keto-acid. In order to determine the qualitative composition of plasma radioactivity, bidimensional thin-layer chromatography of dansyl amino-acids was carried out at 2 and 30min. The results are presented in Table 1. Whole body autoradiography was performed at 5, 10 and 15 min after the intraperitoneal injection of 14C-KG in normal mice. At each time, the presence of labelled compounds was predominantly observed in the intestinal mucosa, liver and parotis salivary glands. Weaker radioactivity was registered in muscle, kidney and the central nervous’system (see a representative result in Figure 2). When the intravenous approach was used, the liver and the kidney showed a higher concentration of radioactivity than the intestinal mucosa (data not shown). In the rats, those organs which showed an accumulation of labelled product in mice were measured for radioactive content 30 min following the intravenous injection of 14C-KG. The results are presented in Figure 3A. The analysis of the labelled amino-acid content was performed using bidimensional micro-TLC in the organs mentioned. These data are summarised in Table 2. When unlabelled a-KG was administered with 14C-KG, there was no modification either in the kinetics of plasma disappearance or in the tissue distribution of the tracer (data not shown). Finally, no significant difference was observed between normal and
+
of “C-x ketoglutarate
+
+
Glutamine + _ _ +
+
to male Wistar
Unidentified +(l
I
+il) +!11 +121
_
burned rats with regard to the labelled-KG plasma decrease (Fig. 1B) and tissue distribution (Fig. 3B). Fate of patentera&-administered
labelled ornithine
The plasma elimination curve following the injection of 14C-Orn is shown in Figure 4A. In healthy rats, a biphasic decrease was observed, also suggesting an open two-compartmental mode1 of elimination for this amino-acid. Following the intraperitoneal administration of 14C-Orn in healthy mice, the highest concentrations of 14C were found in the liver, intestinal mucosa and salivary glands. Radioactivity was also registered in the muscle and kidney. The same results were obtained after intravenous administration. No radioactivity was detected in the central nervous system (Fig. 5). In rats, the quantitative distribution of the tracer in these tissues 30 min after the intra-venous administration of 14C-Om is presented in Figure 6A. The qualitative composition of this radioactivity is shown in Table 3. The presence of unlabelled ornithine did not significantly alter either the rate of plasma elimination of the compound or its tissue distribution (data not shown). In burned animals, the rate of decrease in plasma radioactivity was lower and the kinetic model was more complex than in controls (Fig. 4B). The aspect of the autoradiographs (data not shown) and the amount of radioactivity in the tissues (Fig. 6B) were not modified.
108
METABOLISM
AND
KINETICS
OF PARENTERALLY
ADMINISTERED
ORNITHINE
AND
%-KETOGLUTARATE
- loo
.m z
50
z z
25
z d 12.5
9
z6.25 5 E d 100 E a
a
50
3i a 25 3 125 0 6.25
0
10
20
TIME
rn?!
Fig. 1 Kinetics of plasma elimination of 14C-a ketoglutarate after intravenous injection in normal (A) (n = 6) and burned (B) (n = 6) male Wistar rats.
* ’
DISCUSSION The fate of labelled cc-ketoglutarate in healthy animals After intravenous administration, the kinetics of the plasma elimination of 14C-KG show a rapid biphasic decrease in radioactivity (Fig. IA). Metabolites of a-KG, Glu and Pro, were found in the plasma 30 min after the injection of the compound (Table 1). The rapidity of tissue distribution and the intensity of metabolism observed are not surprising for an endogenous molecule involved in multiple metabolic pathways. These data are consistent with those of a previous study where the metabolism of enterally-administered OKG in healthy men was intensive [ 121. Whole body autoradiography showed that ctKG was present essentially in the intestinal mucosa, liver and salivary glands after intraperitoneal administration (Fig. 2). These data are in accord with those of Servin et al [18] who studied the metabolism of EKG in rats in order to investigate if its psychostimulant effect could be related to the presence of EKG in the central nervous system. Although the concentration of 14C-KG in muscle might appear somewhat low with regard to the data in Figures 2 and 3A, total muscle mass in rats represents approximately 48% of total body weight [ 191 and thus the total muscle-associated radioactivity was probably the highest of all tissues. When the intravenous route was used instead of the peritoneal one, an increase in kidney radioactivity and a fall in intestinal
-
.
. ”
Fig. 2 Tissular distribution of radioactivity 15 min after intraperitoneal administration of 14C-a-keroglutarate in male Swiss mice. Abbreviations: CNS: central nervous system, E: eye, IM: intestinal mucosa, K: kidney, L: liver, M: muscle, SC: salivary glands.
mucosa counts was observed, possibly reflecting the importance of the kidney in metabolising glutamine, glutamate and aKG i.e., in ammoniogenesis [lo]. The analysis of the amino-acid content of the organs was also performed in the rats (Table 2). Three points will be considered: Firstly, labelled glutamate was detected in all selected tissues as was expected from knowledge of amino-acid metabolism and from results obtained after oral administration of calcium crKG to healthy subjects [6]. This amino-acid thus appears to be the major metabolite of administered EKG, reflecting the ubiquitous presence of transaminases. In partictdar, the concomittent presence of 14C-KG and 14C-glutamate in muscle could enable a local synthesis of GL ketoisocaproate from muscular leucine. This branched-chain keto-acid, which possesses an anti-catabolic action [20], could be involved in a muscle-mediated OKG action, as has previously been suggested [9]. Secondly, concerning the presence of labelled glutamine in the tissues studied, it is interesting to note that 14C-glutamine was found both in muscle, which can
CLINICAL NUTRITION
109
L
n Ir L MM
LSGI
Fig. 3 Radioactive content of tissues 30 min after intravenous administration of 14C-a-ketoglutarate to normal (A) (n = 6) and burned (B) (n = 6) male Wistar rats. Abbreviations are as in Figure 2.
produce glutamine from ctKG and Glu, and in organs which use this nitrogen carrier as an energetic substrate, such as the intestine and kidney [lo]. This might indicate that 30 min after 14C-KG administration, inter-organ exchanges and tissular reactions remain very active. Finally, the presence of 14C-glutamate and the absence of 14C-glutamine in the liver are consistent with the notion that hepatic glutamine synthetase and glutaminase activities are both low in the postabsorptive state [lo]. Thirdly, the presence of labelled ornithine and proline in the muscle and intestinal mucosa could arise from labelled glutamate through the intermediary metabolite pyrroline-Scarboxylate (PX), as described by Jones [ 1 l] in the intestinal mucosa of rats. Finally, certain radioactive molecules which were detected but not identified could be related to non-dansylated Krebs cycle intermediaries or urea cycle intermediates. The fate of ornithine in healthy animals The kinetics of plasma elimination of ornithine also gave a biphasic-type curve in healthy rats (Fig. 4A); this
Fig. 4 Kinetics of plasma elimination of 14C-ornithine after intravenous injection in normal (A) (n = 6) and burned (B) (n = 6) male Wistar rats. amino-acid showed rapid and intensive uptake by tissues. Effectively, nearly 93O, of the radioactivity injected had been extracted from the plasma after 30min and at this time several radioactive metabolites or omithine were present in the plasma: glutamate, glutamine, proline and arginine (Table 1). With regard to proline, these results are consistent with the increase in plasma proline found in a previous study [12] after enteral intake of OKG in healthy patients. The labelled products were essentially found in the liver, intestinal mucosa and salivary glands after intraperitoneal administration (Fig. 5). Labelled glutamate and proline were found in all these tissues (except for proline in the intestinal mucosa) and could arise from ornithine by the action of ornithine transaminase and P5C dehydrogenase at the mitochondrial level and P5C reductase in the cytosol [ 111. However, P5C biosynthesis from ornithine has been estimated as 140 times lower in liver than the conversion to citrulline via ornithine transcarbamylase [21]. This concept is consistent with the presence of labelled citrulline in rat liver (Table 3). The metabolism of ornithine in the salivary glands and intestinal mucosa was very intensive since ornithine was totally transformed in 30 min. This could also involve the intermediary metabolite P5C and conversion into either glutamate-glutamine or proline. Furthermore, the biosynthesis of labelled polyamines by omithine decarboxylase was demonstrated in the
110
METABOLISM
AND KINETICS
OF PARENTERALLY
ADMINISTERED
ORNITHINE
AND Z-KETOGLUTARATE
Table 3 Analysis of labelled amino-acids and polyamines content of tissues 30 min after intravenous administration %-ornithine to male Wistar rats Tissue Salivary glands Liver Intestinal mucosa Kidney
Muscle
of
Ornithine
Proline
Glutamate
Glutamine
Citrulline
Polyamines
_
+ + _
_
+
+ + _ _
+(I) _
+
+ + + +
_
+ _
+(3) _
+(3)
+
+
+ ._ _
_
+
_
Unidentified
_
+(2)
1
d
‘P X
A
CNS
K M Fig. 5 Tissular distribution of radioactivity 15 min after intraperitoneal administration of 14C-ornitbine in male Swiss mice. Abbreviations are as in Figure 2.
Fig. 6 Radioactive content of tissues 30 min after intravenous injection of 14C-omithine to normal (A) (n = 6) and burned (B) (n = 6) male Wistar rats. Abbreviations are as in Figure 2.
salivary glands and intestinal mucosa. This ornithineinduced production of polyamines, compounds known to possess anabolic effects [8], could be involved in a mechanism of OKG action at the intestinal level. It is also possible, at least in the case of the salivary glands, that 14C-labelled metabolites had been secondarily transported to this organ.
unlabelled
Orn
In order to approach therapeutic conditions more closely, unlabelled ctKG was added to 14C-KG (and
in plasma
were
ministration
of Orn or aKG,
transport
found.
No modification radioactivity
distribution
Thus,
after
parenteral
the processes
of these two endogenous
in the
or in tissular ad-
of tissular
compounds
were not
saturated. the two
In the light of data from the autoradiographs, components
u-ketoglutarate-ornithine interactions in healthy animals
to 14C-Orn).
rate of decrease
tissue salivary
of the OKG
such as muscle,
molecule liver,
intestinal
glands where these products
gaged
in the
several
cases,
same
metabolic
we observed
appeared
together
mucosa appeared
pathways.
the presence
in
and the to be en-
In effect, of metabolites
in
CLINICAL NUTRITION
common to clKG and Orn, as was the case, for example, for Pro and Glu in muscle. The synergic action of Om and rKG previously suggested by Molimard [22] in the cirrhosis of hyperammonemia in normalisation encephalopathy, thus appears likely and has been confirmed in healthy subjects [6]. Influence
of thermal injury
[61Cynober
171
PI
There was no modification of plasma kinetics and tissular distribution of aKG in burned rats (Figs 1B & 3B). In contrast, thermal injury caused a decrease in the rate of disappearance of plasma 14C-Orn (Fig. 4B), associated with an apparent loss of the bicompartmental model of elimination, although the tissular distribution of labelled ornithine was not altered by the burn state (Fig. 6B). The initial hemodynamic modifications associated with the burn state could be involved in this phenomenon, perhaps with changes in distribution volumes of products or the creation of new equilibrium compartments. On the other hand, it is likely that 14Cornithine uptake by organs is lower in burned rats, either at the membrane carrier level, or at the level of metabolising enzymes, whose synthesis is probably diminished, protein synthesis being reduced after bum injury [23].
[91
[lOI
[Ill
1121
[I31
[I41 ACKNOWLEDGEMENTS for her expert technical assistof Pharmacology, University Paris XI, Professor Jacquot) for making the equipment required for autoradiography available to us. Thanks also fo MS Pascale Jue for valuable secretarial help. This work was supported in part by grants from Laboratoires J. Logeais, Issy les Moulineaux 92130 France. We thank
Mrs Solange
Poujol
[I51
ance and Dr Payent (Department
[lb1
[I71 REFERENCES 1181
[l] Leander
[2]
[3]
[4]
[5]
U, Fiirst P, Vesterberg K, Vinnars E, 1985 Nitrogen sparing effect of Ornicetil in the immediate postoperative state. I-Clinical biochemistry and nitrogen balance. Clinical Nutrition 4: 43-51 Cynober L, Saizy R, Nguyen Dinh F, Lioret N, Giboudeau J 1984 Effect of enterally administered ornithine a ketoglutarate on plasma and urinary amino acid levels after burn injury. Journal of Trauma 24: 59&596 Demarcq J M, Delbar M, Trochu G, Crignon J J 1984 Effets de l’alpha cetoglutarate d’omithine sur l’btat nutritionnel des malades de rbnimation. Cahiers Anesthisiologie 32: 3-6 Cynober L, Blonde F, Lioret N, Coudray-Lucas C, Saizy R, Giboudeau J 1986 Arterio-venous differences in amino acids, glucose, lactate and fatty acids in bum patients; effect of ornithine alpha-ketoglutarate. Clinical Nutrition 5: 221-226 Nicolas F, Rodineau P 1982 Essai contr8li: croise de I’alpha cetoglutarate d’omithine en alimentation em&ale. Ouest Med 35: 711-713
Submission date: 3 February
1987. Accepted: 3 November
1987.
[I91
rJo1
Pll
1221
1231
111
L, Aussel C, Coudray-Lucas C et al 1986 Action of omithine a-ketoglutarate, omithine chlorhydrate and calcium a-ketoglutarate on plasma amino acid and hormonal patterns in healthy subjects. Clinical Nutrition suppl5: abstract Vaubourdolle M, Lioret N, Cynober L, Aussel C, Coudray-Lucas C, Saizy R, Giboudeau J 1985 Influence of enterally administered ornithine a ketoglutarate on hormonal patterns in burn patients. Burns 13: 349-356 Grill0 M A 1985 Metabolism and function of polyamines. International Journal of Biochemistry 17: 945948 Aussel C, Cynober L, Lioret N, Dubreuil M, Saizy R, Giboudeau J 1986 Branched-chain keto acids plasma concentrations following bum injury. American Journal of Clinical Nutrition 44: 825-831 Souba W W, Smith R J, Wilmore D W 1985 Glutamine metabolism by the intestinal tract. Journal of Parenteral and Enteral Nutrition 9: 608-617 Jones M E 1985 Conversion of glutamate to ornithine and proline: pyrroline-5-carboxylate, a possible modulator of arginine requirements. Journal of Nutrition 115: 509-5 15 Cynober L, Vaubourdolle M, Dore A, Giboudeau J 1984 Kinetics and metabolic effects of orally administered ornithine a-ketoglutarate in healthy subjects fed with 2 standardized regimen. American Journal of Climcal Nutrition 39: 514-519 Eriksson L S, Reihner E, Wahren J 1985 Infusion of ornithine-alpha-ketoglutarate in healthy subjects: effects on protein metabolism. Clinical Nutrition 4: 73-76 Turinsky J 1982 Regional differences in in vivo skeletal muscle responsiveness to insulin after burn injury. Experimental and Molecular Pathology 37: 2 17-224 Biou D, Queyrel N, Visseaux M N, Collignon I, Pays M 1981 Separation and identification of dansylated human serum and urinary amino acid by two-dimensional &in-layer chromatography. Journal of Chromatography 226: 477-483 Cohen Y, Delassue H 1959 Modification de la methode d’autoradiographie de S Ullberg sur coupes de souris entieres. CR Sot Biol 153: 300-304 Labaune J P 1984 In: Pharmacocinetique: principes fondamentaux. Masson, Paris, pp 206-223 Servin A, Lindenbaum A, Skeretkowicz J, Wepierre J 1981 Pharmacocinbtique et m&abolisme de l’acide z-cbtoglutarique chez l’animal. In: Experimental Pharmacokinetics, Vol. 2. Technique et Documentation, Paris pp 178-186 Deno D L, Lewis F P, Saba T M 1984 Clearance from the vascular compartment of endogenous by labelled plasma fibronectin. Journal of Trauma 24: 91-98 Walser M 1983 Rationale and indications for the use of z-keto analogues. Journal of Parenteral and Enteral Nutrition 8: 37-41 Sochor M, McLean P, Brown J, Greenbaum A L. 1981 Regulation of pathways of ornithine metabolism. Enzyme 26: 15-23 Molimard R, Charpentier C, Lemonnier F 1982 Modifications de l’aminoacidtmie des cirrhotiques sous l’influence de sels d’omithine. Annals of Nutrition and Metabolism 26: 25-36 Frayn K N, Maycock P F 1979 Regulation of protein metabolism by a physiological concenrration of insulin in mouse soleus and extensor digitorum longus muscles. Biochemistry Journal 184: 323-330
NUTRITION , Longman
(19.W) 7: 105-l 11 I.rd 1988
Group IJK
Metabolism and Kinetics of Parenterally Administered Ornithine and a-Ketoglutarate in Healthy and Burned Animals M. Vaubourdolle*, and L. Cynober* *Laboratoire
de Biochimie
A. Jardelt, CNRS
C. Coudray-Lucas*,
UA 622 and tLaboratoire
de Physiologie,
92296 Chatenay Malabry Cedex, France (Reprint requeststo M.V.)
0. G. Ekindjian*, Universitk
J. Agneray*
Paris XI, Tour D4,5
Rue JB Clkment,
To improve our understanding of the metabolism of ornithine a-ketoglutarate (OKG), ABSTRACT used as an adjuvant of parenteral nutrition, we studied the plasma kinetics, localisation of target tissues and metabolism of a ketoglutarate (aKG) and ornithine (Orn) in healthy and burned animals. After parenteral administration, the kinetics of plasma disappearance of the two labelled compounds showed a biphasic decrease reflecting an open two-compartmental model of elimination, with the exception of Orn in burned rats. The appearance of metabolites in the plasma was rapid, particularly with regard to glutamate, proline and, following ornithine administration, glutamine. This witnessed the engagement of the substrates in multiple metabolic pathways. The study of tissular distribution by autoradiography demonstrated certain target tissues in common for aKG and Orn such as the liver, intestinal mucosa, salivary glandsj kidney and muscle. This is consistent with a possible synergic action of these two compounds. The identification of labelled amino-acids and aliphatic polyamines was also performed in the tissues. Two major observations were made, which could be of interest in further work concerning cellular mechanisms of OKG action. Firstly, after aKG administration, labelled Glu and aKG were detected simultaneously in muscle; this would render possible the local biosynthesis of the anticatabolic compound a ketoisocaproate. Secondly, the injection of 14C-ornithine was followed by the appearance in the intestinal mucosa and salivary glands of labelled aliphatic polyamines, which possess an anabolic effect.
INTRODUCTION Ornithine r-ketoglutarate (OKG) has been used in the
treatment of traumatised patients as an adjuvant in parenteral and enteral nutrition with anabolic and/oranticatabolic effects [14]. These effects include a rapid improvement in the nitrogen balance [ 1, 3-51, a reduction in hyperphenylalaninemia [2] and an improvement in plasma levels of visceral proteins [3]. However, many questions concerning this compound remain unresolved. Firstly, although there are numerous in vitro studies on isolated cells or organs concerning ornithine and rKG metabolism, to our knowledge little work has been don: on the in vivo metabolism of administered Om and/or rKG. The only existing study performed in humans involved the oral administration of Om, clKG and OKG to healthy subjects [6]. An increase in plasma glutamate levels after OKG, aKG, and Om loads was observed, whereas Pro and Arg increased only after the OKG load. However, plasma amino-acid variations is the net re-
sult of interorgan exchanges. It thus appeared desirable to perform animal experiments in order to determine the role of the various organs in the metabolism of Orn and aKG administered in vivo. Secondly, the mechanism of OKG action is unknown. Although it has been postulated [l-2] that OKG exerts its anabolic/anticatabolic action via a stimulation of insulin or growth hormone secretion in traumatised patients, this hypothesis remains debatable [7]. Other possible mechanisms might involve either the formation of polyamines from ornithine [8] or the production of branched-chain ketoacids in the transamination reaction involving a-ketoglutarate (aKG) [9], or further the production of glutamine from both ornithine and aKG [lo]. In effect, ornithine and aKG possess partially common metabolic pathways [ 1 l] and, when administered simultaneously, the repercussions of each compound on the metabolism of the other are unknown. Another interesting point remains obscure: OKG administered to surgical [I] and bum patients [2] leads to abnormally high plasma and muscle concentrations of omithine and proline, its metabolite, compared
105
106
METABOLISM AND KINETICS OF PARENTERALLY
ADMINISTERED ORNITHINE AND 1-KETOGLUTARATE
to values obtained in experiments on healthy subjects [ 12, 131. The reasons for this accumulation of ornithine remain unclear. In order to investigate the metabolism of OKG, before going on to study to its mechanism of action, we studied here the plasma disappearance, target tissues and metabolites of the two components of the OKG molecule in healthy and burned animals using the parenteral route of administration.
MATERIALS
AND
METHODS
Kinetics and metabolic studies Male Wistar rats weighing 200-300g were fed a commercial pelleted diet (A03 regimen, UAR) ad libitum. At the beginning of the experiments, half the animals were anaesthetised with diethylether and burned by immersing the dorsum in water at 80°C for 10 s, producing a full-thickness scald injury over 20% of the body surface area [ 141. A volume of 0.15 M NaCl, corresponding to 1076 of the body weight, was administered parenterally in order to prevent any eventual state of shock [14]. The burned rats showed no sign of discomfort and did not receive pain medication. Three days following the thermal injury, all the rats were starved for 3 h and anaesthetised with thiopental (25mg/kg). The animals were cannulated in the right external jugular vein for intravenous injection and a catheter was placed in the left carotid artery for blood sampling. Then at (to), 25uCi of a-[U-14Qketoglutarate (14C-KG, spec. act. 258 mCi/mmol, NEN, Albany, USA) or 25 uCi of [U-14C]-ornithine (14C-Om, spec, act, 266 mCi/mmol, NEN, Albany, USA) was administered parenterally (volume 0.5ml) to the healthy and burned rats. Certain animals received 14C- KG (or 14C-Orn), diluted in a solution of unlabelled c1KG (or Orn) at a concentration corresponding to 0.5g of ornithine cr-ketoglutarate per kg of body weight, a dose used in human therapeutics [2]. Venous blood (0.25 ml) was collected in heparinised tubes at 0.5, 1, 2, 5, 10, 15 and 30 min after parenteral injection; the radioactivity present in 0.1 ml of plasma was measured in 5ml aqualuma solvent with a liquid scintillation counter. In some cases (see below), 0.1 ml of plasma was stored at -20°C for qualitative analysis of radioactivity. At the linal time (30 min), the rats were killed by decapitation and the liver, kidneys, salivary glands, soleus muscle and intestinal mucosa were collected. After weighing, each piece of tissue was homogenised at 4°C in 0.3M perchloric acid containing 0.5 mM EDTA (5 ml/g of tissue). After centrifugation (12 000 g, 15 min) and measurement of the total volume, the supematant was removed and divided into several
aliquots for the determination of total radioactivity and the qualitative analysis of labelled amino-acids. For the latter, bidimensional thin-layer chromatography (TLC) of dansylated amino-acids was carried out as described by Biou et al [15] on micropolyamide plates (F 1700, 5 x 5cm, Schleicher and Schiill, Dassel, FRG). The fluorescent spots were cut out and dissolved in 5ml scintillation fluid for counting. Amino-acids were identified by calculating the position of the spot, with reference to dansyl-glycine. The initial method was applicable for the detection and identification of labelled aliphatic polyamines (putrescine, spermine, spermidine). The minimal detectable amount was in the picomole range.
Whole body autoradiography Male Swiss mice weighing 20-22g were used for the experiments. The nutrition and thermal injury were as described above. On the day of the experiment, 25 uCi of 14C-KG or 14C-Orn, with or without unlabelled KG or Orn respectively, was administered parenterally (see above). Five, 10 and 15min following peritoneal injection or 2 and 5 min after intravenous administration, the mice were killed by immersion in acetone at -70°C and stored at -20°C for 1 week. Sagittal sections of the animals, 20 urn thick, were prepared with a microtome and dried at -20°C. The sections were placed on X-O- mat film (Kodak) for 3-4 weeks as described by Cohen and Delassue [ 161 and then developed. There were three animals in each of the above series, i.e., presence or absence of burns, addition or not of unlabelled compounds, peritoneal or intravenous administration.
Statistics Data are given as mean & SD. Comparisons were made by the Mann Whitney U-test and differences with p values of less than 0.05 were considered significant. The statistical analysis of the kinetics of plasma radioactivity was carried out by the method of residuals [ 171 and linear regression.
RESULTS Fate ofparenterally-administered labelled u-ketoglutarate The decrease in plasma radioactivity as a function of time after jugular injection of 14C-KG in healthy rats (n=6) is presented in Figure 1A. The experimental curve can be superimposed on the graphic representation of the equation: y = Ae + Be. This may reflect the existence of an open two-compartmental model for the
CLINICAL
Table 1 rats
Analysis
of plasma
radioactivity
%-ctKetoglutarate “C-ornithine
tcpm
*cpm > 800;, of total.
Analysis
Tissue Kidney Liver Salivary glands Intestinal mucosa
Muscle
of labelled
content
3~Ketoglutarate + + + +
+
injection
of labelled
Labelled
Time after injection (min)
Administered compound
Table 2 rats
after intravenous
products
2 30
+ +*
_ _
2 30
_ _
+t
or Orn in male Wistar
recovered Unidentified molecules
Amino-acids
Ornithine
a Ketoglutarate
rKG
_
I
Glutamate Proline _
+
107
NUTRITION
_. 1
Glutamate Glutamine Proline Arginine
> 600/, of total.
of tissues 30min
after intravenous
Ornithine
Proline
administration
Glutamate
_
_
_
_
+
-
_
+
+
+ +
+
elimination of this keto-acid. In order to determine the qualitative composition of plasma radioactivity, bidimensional thin-layer chromatography of dansyl amino-acids was carried out at 2 and 30min. The results are presented in Table 1. Whole body autoradiography was performed at 5, 10 and 15 min after the intraperitoneal injection of 14C-KG in normal mice. At each time, the presence of labelled compounds was predominantly observed in the intestinal mucosa, liver and parotis salivary glands. Weaker radioactivity was registered in muscle, kidney and the central nervous’system (see a representative result in Figure 2). When the intravenous approach was used, the liver and the kidney showed a higher concentration of radioactivity than the intestinal mucosa (data not shown). In the rats, those organs which showed an accumulation of labelled product in mice were measured for radioactive content 30 min following the intravenous injection of 14C-KG. The results are presented in Figure 3A. The analysis of the labelled amino-acid content was performed using bidimensional micro-TLC in the organs mentioned. These data are summarised in Table 2. When unlabelled a-KG was administered with 14C-KG, there was no modification either in the kinetics of plasma disappearance or in the tissue distribution of the tracer (data not shown). Finally, no significant difference was observed between normal and
+
of “C-x ketoglutarate
+
+
Glutamine + _ _ +
+
to male Wistar
Unidentified +(l
I
+il) +!11 +121
_
burned rats with regard to the labelled-KG plasma decrease (Fig. 1B) and tissue distribution (Fig. 3B). Fate of patentera&-administered
labelled ornithine
The plasma elimination curve following the injection of 14C-Orn is shown in Figure 4A. In healthy rats, a biphasic decrease was observed, also suggesting an open two-compartmental mode1 of elimination for this amino-acid. Following the intraperitoneal administration of 14C-Orn in healthy mice, the highest concentrations of 14C were found in the liver, intestinal mucosa and salivary glands. Radioactivity was also registered in the muscle and kidney. The same results were obtained after intravenous administration. No radioactivity was detected in the central nervous system (Fig. 5). In rats, the quantitative distribution of the tracer in these tissues 30 min after the intra-venous administration of 14C-Om is presented in Figure 6A. The qualitative composition of this radioactivity is shown in Table 3. The presence of unlabelled ornithine did not significantly alter either the rate of plasma elimination of the compound or its tissue distribution (data not shown). In burned animals, the rate of decrease in plasma radioactivity was lower and the kinetic model was more complex than in controls (Fig. 4B). The aspect of the autoradiographs (data not shown) and the amount of radioactivity in the tissues (Fig. 6B) were not modified.
108
METABOLISM
AND
KINETICS
OF PARENTERALLY
ADMINISTERED
ORNITHINE
AND
%-KETOGLUTARATE
- loo
.m z
50
z z
25
z d 12.5
9
z6.25 5 E d 100 E a
a
50
3i a 25 3 125 0 6.25
0
10
20
TIME
rn?!
Fig. 1 Kinetics of plasma elimination of 14C-a ketoglutarate after intravenous injection in normal (A) (n = 6) and burned (B) (n = 6) male Wistar rats.
* ’
DISCUSSION The fate of labelled cc-ketoglutarate in healthy animals After intravenous administration, the kinetics of the plasma elimination of 14C-KG show a rapid biphasic decrease in radioactivity (Fig. IA). Metabolites of a-KG, Glu and Pro, were found in the plasma 30 min after the injection of the compound (Table 1). The rapidity of tissue distribution and the intensity of metabolism observed are not surprising for an endogenous molecule involved in multiple metabolic pathways. These data are consistent with those of a previous study where the metabolism of enterally-administered OKG in healthy men was intensive [ 121. Whole body autoradiography showed that ctKG was present essentially in the intestinal mucosa, liver and salivary glands after intraperitoneal administration (Fig. 2). These data are in accord with those of Servin et al [18] who studied the metabolism of EKG in rats in order to investigate if its psychostimulant effect could be related to the presence of EKG in the central nervous system. Although the concentration of 14C-KG in muscle might appear somewhat low with regard to the data in Figures 2 and 3A, total muscle mass in rats represents approximately 48% of total body weight [ 191 and thus the total muscle-associated radioactivity was probably the highest of all tissues. When the intravenous route was used instead of the peritoneal one, an increase in kidney radioactivity and a fall in intestinal
-
.
. ”
Fig. 2 Tissular distribution of radioactivity 15 min after intraperitoneal administration of 14C-a-keroglutarate in male Swiss mice. Abbreviations: CNS: central nervous system, E: eye, IM: intestinal mucosa, K: kidney, L: liver, M: muscle, SC: salivary glands.
mucosa counts was observed, possibly reflecting the importance of the kidney in metabolising glutamine, glutamate and aKG i.e., in ammoniogenesis [lo]. The analysis of the amino-acid content of the organs was also performed in the rats (Table 2). Three points will be considered: Firstly, labelled glutamate was detected in all selected tissues as was expected from knowledge of amino-acid metabolism and from results obtained after oral administration of calcium crKG to healthy subjects [6]. This amino-acid thus appears to be the major metabolite of administered EKG, reflecting the ubiquitous presence of transaminases. In partictdar, the concomittent presence of 14C-KG and 14C-glutamate in muscle could enable a local synthesis of GL ketoisocaproate from muscular leucine. This branched-chain keto-acid, which possesses an anti-catabolic action [20], could be involved in a muscle-mediated OKG action, as has previously been suggested [9]. Secondly, concerning the presence of labelled glutamine in the tissues studied, it is interesting to note that 14C-glutamine was found both in muscle, which can
CLINICAL NUTRITION
109
L
n Ir L MM
LSGI
Fig. 3 Radioactive content of tissues 30 min after intravenous administration of 14C-a-ketoglutarate to normal (A) (n = 6) and burned (B) (n = 6) male Wistar rats. Abbreviations are as in Figure 2.
produce glutamine from ctKG and Glu, and in organs which use this nitrogen carrier as an energetic substrate, such as the intestine and kidney [lo]. This might indicate that 30 min after 14C-KG administration, inter-organ exchanges and tissular reactions remain very active. Finally, the presence of 14C-glutamate and the absence of 14C-glutamine in the liver are consistent with the notion that hepatic glutamine synthetase and glutaminase activities are both low in the postabsorptive state [lo]. Thirdly, the presence of labelled ornithine and proline in the muscle and intestinal mucosa could arise from labelled glutamate through the intermediary metabolite pyrroline-Scarboxylate (PX), as described by Jones [ 1 l] in the intestinal mucosa of rats. Finally, certain radioactive molecules which were detected but not identified could be related to non-dansylated Krebs cycle intermediaries or urea cycle intermediates. The fate of ornithine in healthy animals The kinetics of plasma elimination of ornithine also gave a biphasic-type curve in healthy rats (Fig. 4A); this
Fig. 4 Kinetics of plasma elimination of 14C-ornithine after intravenous injection in normal (A) (n = 6) and burned (B) (n = 6) male Wistar rats. amino-acid showed rapid and intensive uptake by tissues. Effectively, nearly 93O, of the radioactivity injected had been extracted from the plasma after 30min and at this time several radioactive metabolites or omithine were present in the plasma: glutamate, glutamine, proline and arginine (Table 1). With regard to proline, these results are consistent with the increase in plasma proline found in a previous study [12] after enteral intake of OKG in healthy patients. The labelled products were essentially found in the liver, intestinal mucosa and salivary glands after intraperitoneal administration (Fig. 5). Labelled glutamate and proline were found in all these tissues (except for proline in the intestinal mucosa) and could arise from ornithine by the action of ornithine transaminase and P5C dehydrogenase at the mitochondrial level and P5C reductase in the cytosol [ 111. However, P5C biosynthesis from ornithine has been estimated as 140 times lower in liver than the conversion to citrulline via ornithine transcarbamylase [21]. This concept is consistent with the presence of labelled citrulline in rat liver (Table 3). The metabolism of ornithine in the salivary glands and intestinal mucosa was very intensive since ornithine was totally transformed in 30 min. This could also involve the intermediary metabolite P5C and conversion into either glutamate-glutamine or proline. Furthermore, the biosynthesis of labelled polyamines by omithine decarboxylase was demonstrated in the
110
METABOLISM
AND KINETICS
OF PARENTERALLY
ADMINISTERED
ORNITHINE
AND Z-KETOGLUTARATE
Table 3 Analysis of labelled amino-acids and polyamines content of tissues 30 min after intravenous administration %-ornithine to male Wistar rats Tissue Salivary glands Liver Intestinal mucosa Kidney
Muscle
of
Ornithine
Proline
Glutamate
Glutamine
Citrulline
Polyamines
_
+ + _
_
+
+ + _ _
+(I) _
+
+ + + +
_
+ _
+(3) _
+(3)
+
+
+ ._ _
_
+
_
Unidentified
_
+(2)
1
d
‘P X
A
CNS
K M Fig. 5 Tissular distribution of radioactivity 15 min after intraperitoneal administration of 14C-ornitbine in male Swiss mice. Abbreviations are as in Figure 2.
Fig. 6 Radioactive content of tissues 30 min after intravenous injection of 14C-omithine to normal (A) (n = 6) and burned (B) (n = 6) male Wistar rats. Abbreviations are as in Figure 2.
salivary glands and intestinal mucosa. This ornithineinduced production of polyamines, compounds known to possess anabolic effects [8], could be involved in a mechanism of OKG action at the intestinal level. It is also possible, at least in the case of the salivary glands, that 14C-labelled metabolites had been secondarily transported to this organ.
unlabelled
Orn
In order to approach therapeutic conditions more closely, unlabelled ctKG was added to 14C-KG (and
in plasma
were
ministration
of Orn or aKG,
transport
found.
No modification radioactivity
distribution
Thus,
after
parenteral
the processes
of these two endogenous
in the
or in tissular ad-
of tissular
compounds
were not
saturated. the two
In the light of data from the autoradiographs, components
u-ketoglutarate-ornithine interactions in healthy animals
to 14C-Orn).
rate of decrease
tissue salivary
of the OKG
such as muscle,
molecule liver,
intestinal
glands where these products
gaged
in the
several
cases,
same
metabolic
we observed
appeared
together
mucosa appeared
pathways.
the presence
in
and the to be en-
In effect, of metabolites
in
CLINICAL NUTRITION
common to clKG and Orn, as was the case, for example, for Pro and Glu in muscle. The synergic action of Om and rKG previously suggested by Molimard [22] in the cirrhosis of hyperammonemia in normalisation encephalopathy, thus appears likely and has been confirmed in healthy subjects [6]. Influence
of thermal injury
[61Cynober
171
PI
There was no modification of plasma kinetics and tissular distribution of aKG in burned rats (Figs 1B & 3B). In contrast, thermal injury caused a decrease in the rate of disappearance of plasma 14C-Orn (Fig. 4B), associated with an apparent loss of the bicompartmental model of elimination, although the tissular distribution of labelled ornithine was not altered by the burn state (Fig. 6B). The initial hemodynamic modifications associated with the burn state could be involved in this phenomenon, perhaps with changes in distribution volumes of products or the creation of new equilibrium compartments. On the other hand, it is likely that 14Cornithine uptake by organs is lower in burned rats, either at the membrane carrier level, or at the level of metabolising enzymes, whose synthesis is probably diminished, protein synthesis being reduced after bum injury [23].
[91
[lOI
[Ill
1121
[I31
[I41 ACKNOWLEDGEMENTS for her expert technical assistof Pharmacology, University Paris XI, Professor Jacquot) for making the equipment required for autoradiography available to us. Thanks also fo MS Pascale Jue for valuable secretarial help. This work was supported in part by grants from Laboratoires J. Logeais, Issy les Moulineaux 92130 France. We thank
Mrs Solange
Poujol
[I51
ance and Dr Payent (Department
[lb1
[I71 REFERENCES 1181
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