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Increased kynurenic acid in the brain after neonatal asphyxia
Life Sciences 69 (2001) 1249–1256
Increased kynurenic acid in the brain after neonatal asphyxia H. Barana,*, B. Kepplingerb, M. Herrera-Marschitzc, K. Stolzea, G. Lubecd, H. Nohla a
Institute of Pharmacology and Toxicology, Veterinary Medical University Vienna, A-1210 Vienna, Austria b Department of Neurology, Diagnostic and Therapy Center LNK, A-3362 Mauer/Amstetten, Austria c Department of Physiology and Pharmacology, 17177 Stockholm, Sweden d Department of Pediatrics, University of Vienna, A-1090 Vienna, Austria Received 17 October 2000; accepted 8 February 2001
Abstract
In the brain, L-kynurenine is an intermediate for the formation of kynurenic acid, a metabolite with neuroprotective activities, and a substrate for the synthesis of 3-hydroxy-kynurenine, a metabolite with neurotoxic properties. In the present study, alterations of L-kynurenine, 3-hydroxy-kynurenine and kynurenic acid levels were examined in the brain of neonatal (10 minutes old) rats after 5, 10, 15 or 20 minutes of asphyxia, and in the brain of the corresponding caesarean-delivered controls, using sensitive high-performance liquid chromatographic methods. Among kynurenines we found a marked time-dependent increase of kynurenic acid levels, a moderately delayed increase of 3-hydroxy-kynurenine, and a trend for a decrease of L-kynurenine content. Thus, the brain reacted rapidly to the oxygen deficit by increasing kynurenic acid levels by 44 % already after 5 minutes of asphyxia, and the most prominent elevation of kynurenic acid (302 % of control) was found after 20 minutes of asphyxia - the critical time limit of survival. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Asphyxia; Excitatory amino acids; Kynurenic acid; Neuroprotection; Mitochondria, Lethality
Introduction Perinatal asphyxia is considered to lead to a variety of pathological conditions and may be linked to psychiatric and neurologic disorders later in life. Experimental data suggest a role of excitatory amino acids (EAA) in the genesis of ischemic damage in the immature brain * Corresponding author. Institute of Pharmacology and Toxicology, Veterinary University Vienna, A-1210 Vienna, Austria. E-mail address: [email protected] (H. Baran) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 1 5 -2
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[1]. It has been shown that the exogenous excitatory amino acids e.g. quisqualate and N-methyl-D-aspartate (NMDA) are potent excitotoxins in the immature brain [1]. Glutamate and aspartate levels are significantly elevated in the brain during hypoxic-ischemia of immature lambs [2] and rats [30]. Experimental animal studies have shown that the non-competitive NMDA receptor blocker MK-801 attenuates brain damage induced by hypoxic-ischemic insults [3], and kynurenic acid, a well known antagonist of EAA receptors with a high affinity to the glycine site of the NMDA receptor complex [4], has been used as neuroprotectant and anticonvulsant in various experimental conditions [4, 5]. L-kynurenine and kynurenic acid levels have been found to be development-dependent. Thus, L-kynurenine and kynurenic acid levels decline rapidly after birth [6, 7]. Since it has been shown in 7 days old neonatal rats that kynurenic acid offered a partial neuroprotection after hypoxic-ischemia [8], we questioned the possibility of kynurenine metabolism alteration in the asphyctic brain. In addition, we were interested on investigating another metabolite of L-kynurenine, 3-hydroxykynurenine, which declines after birth [7], but may display neuronal cytotoxicity [4]. Thus, the aim of present work was to study the endogenous levels of L-kynurenine, kynurenic acid and 3-hydroxy-kynurenine in the rat brain immediately after birth (10 minutes of age) following different degrees of perinatal asphyxia. A preliminary account of this work has been presented elsewhere [9]. Methods Materials L-kynurenine, kynurenic acid and 3-hydroxy-kynurenine were purchased from Sigma. All other chemicals used were also of the highest commercially available purity. Animal experiments Asphyxia was induced in pups delivered by a Caesarean section on pregnant SpragueDawley rats as described earlier [10]. In the present model, rats within the last day of gestation (as evaluated by protocols from the Animal Department), were anaesthetized with ether and hysterectomised. The uterus horns, still containing the fetuses were taken out, and placed into a water bath at 378C for various periods of time (5 to 20 minutes). Immediately after hysterectomy one or two pups were delivered to be considered caesarean-delivered (0 min asphyxia) controls, before the uterus horns with the remaining fetuses were placed into the water bath. Thus, control and asphyctic pups were obtained from the same mother, since each rat provided approximately 10–14 pups. After the corresponding asphyctic periods at 378C, the uterus horns were rapidly opened and the pups were removed and stimulated to breathe by cleaning the amniotic fluid and tactile stimulation of the oral region with pieces of medical wipes. The pups were intensively stimulated for approximately 5 minutes until a gasp was produced, or a decision was made that the pups were unable to recover. Further intermittent stimulation was performed on a heating pad to be sure that regular breathing was established, and thereafter killed by decapitation 10 minutes after the corresponding asphyctic periods. Control pups were decapitated 10 minutes after caesarean-delivery. The brain (cerebrum and cerebellum) immediately was removed and stored at 2808C pending further analysis. The
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animal studies were approved by the local Ethical Committee (Stockholm’s Norra Djurförsöksetiska Nämnd). Tissue preparation and neurochemical analysis Brains were homogenized by a Potter followed by a Polytron (Kinematics GmbH, Luzern, Switzerland) on an ice bath in 5 volumes (wt/vol) of 0.1 M HCl. The homogenates were centrifuged at 18,000 rpm for 15 min at 48C and one ml of the supernatant was applied to a Dowex 50 W cation exchange column pre-washed with 0.1 M HCl. Thereafter, the column was washed again with 1 ml 0.1 M HCl and 1 ml distilled water, and kynurenic acid was eluted with 2 ml distilled water [11]. L-kynurenine and 3-hydroxy-kynurenine were eluted with 2 ml of 1 M NH4OH [12]. The aliquots were frozen, lyophilized and then stored at 2308C until analysis. Prior to analysis the samples were re-suspended in 250 ml of distilled water for kynurenic acid, or in 250 ml of 0.1 M HCl for L-kynurenine and 3-hydroxy-kynurenine measurements, respectively. Kynurenic acid was measured by high performance liquid chromatography (HPLC) coupled to a fluorescence detection system, as previously reported [13]. The HPLC system used for analysis of kynurenic acid consisted of the following: a pump (Shimadzu, LC-6A), a fluorescence detector (Shimadzu, RF-535) set at an excitation wavelength of 340 nm, an emission wavelength of 398 nm, and a Shimadzu C-R5A Chromatopac Integrator. The mobile phase (isocratic system) consisted of 50 mM sodium acetate, 250 mM zinc acetate and 4 % acetonitrile, pH 6.2, and was pumped through a 10 cm 3 0.4 cm column (HR-80, C-18, particle size 3 mM, InChrom, Austria) at a flow rate of 1.0 ml/min, run at room temperature (238C). L-kynurenine and 3-hydroxy-kynurenine were measured by HPLC coupled with UV detector at 365 nm. The mobile phase contained 0.1 M ammonium acetate, 0.1 M acetic acid, and 2 % acetonitrile [14]. Protein determination Protein was measured according to the method of Bradford [15] using a commercially available kit (BIO-RAD) and bovine serum albumin as a standard. Statistics Kynurenine metabolite levels are per mg of protein. In addition we calculated the rate of kynurenic acid formation during the last 5 minutes for each period of asphyxia and expressed it as rate per minute. All data are given as means 6 S.E.M. For comparison of groups the Student t-test or Mann Whitney U-Test were applied. The level for statistical significance was p,0.05. Results In line with a prior study [10] we observed a progressive survival decline, in parallel with an increase of asphyxia time in perinatal pups. Until 15 minutes of asphyxia 100 % survival was observed. Survival declined to a 60 % after 20 minutes of asphyxia. We observed a moderate decrease of L-kynurenine content in the brain of asphyctic pups from 5 to 20 minutes of asphyxia (Table 1). Measurement of kynurenic acid revealed a significant and marked increase after all periods of asphyxia. Following 5 minutes of asphyxia the concentration of kynurenic acid was increased already by 44 % followed by 77 % after 10,
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Table 1 Kynurenine metabolites in the rat brain after different times of asphyxia Group (time of asphyxia)
L-kynurenine (pmol/mg protein)
Kynurenic acid (fmol/mg protein)
3-Hydroxykynurenine (fmol/mg protein)
13.09 6 0.69 (100 %) 11.75 6 0.68 (90 %) 11.63 6 1.17 (89 %) 12.22 6 0.73 (93 %) 11.31 6 1.08 (86 %)
335.86 17.1 (100 %) 482.29 653.1* (144 %) 593.6 6 59.0** (177 %) 726.0 6 73.3*** (216 %) 1013.7 6 62.6*** (302 %)
716.2 6 61.2 (100 %) 624.8 6 49.2 (87 %) 673.9 6 36.0 (94 %) 770.1 6 66.2 (108 %) 858.3 6 54.0* (120 %)
0 minutes (control) 5 minutes 10 minutes 15 minutes 20 minutes
Data represent a mean 6 S.E.M. of 10 control and 9–10 asphyxia animals. In parenthesis, % of control. Significances by using Mann Whitney U-Test: * p,0.05; ** p,0.01; *** p,0.01 vs control.
116 % after 15 and 202 % after 20 minutes, as compared to the controls. The highest rate of kynurenic acid formation during the last 5 minutes for each asphyxia period occurred after 20 minutes of asphyxia (Table 2). 3-Hydroxy-kynurenine declined moderately after 5 minutes of asphyxia, to be then normalized after 10, and moderately, but significant increased after 20 minutes of asphyxia.
Discussion Perinatal asphyxia is a major cause of death or neurological injury in newborn infants. In experimental animals it has been shown that the number of surviving pups declined significantly with an increase of the length of asphyxia [10]. In line with that study a 100 % of survival was observed up to 15 minutes of asphyxia, while only 60 % of the pups survived after 20 minutes of asphyxia. Asphyxia for longer than 21 minutes at 378C led to 100 % of mortality [10]. Among the investigated kynurenines e.g. L-kynurenine, kynurenic acid and 3-hydroxykynurenine, only kynurenic acid was markedly altered after asphyxia. The brain reacted rapidly to oxygen deficit (already after 5 minutes of asphyxia) by increasing kynurenic acid levels. The elevation of kynurenic acid levels was time-dependent. The rate of kynurenic acid elevaTable 2 Rate of kynurenic acid formation in the brain after different periods of asphyxia Rate of kynurenic acid formation 5 Minutes periods Kynurenic acid [fmol/mg protein/minute]
0–5 minutes
5–10 minutes
10–15 minutes
15–20 minutes
29.3 6 3.2
22.3 6 2.2
26.5 6 2.7
57.5 6 3.6***
Kynurenic acid formation rate was integrated for the last 5 minutes of each asphyxia period and calculated per minute. Data represent a mean 6 S.E.M. of 9–10 animals. Statistic significance was tested with the Student t-test: *** P , 0.001 versus the first 5 minutes-period.
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tion calculated per minute within 5 minute-periods was remarkably stable until 15 minutes of asphyxia, whereas after 20 minutes i.e. within the 4th 5 minute-period, the rate suddenly doubled. This indicates that mortality of asphyctic pups coincided with the highest rate of endogenous kynurenic acid formation. Interestingly, the evaluation of adult rats behaviour after intraventricular kynurenic acid application revealed dose-dependent changes, including stereotypy, ataxia, sleep and death, respectively [16]. On the other hand, intraperitoneally applied kynurenic acid offered a partial neuronal protection for hypoxic-ischemia in 7 days old neonatal rats, however the respiratory frequency and behaviour of the animals did not differ among treated and non-treated hypoxic-ischemia animals [8]. At present, there is no direct evidence that high kynurenic acid levels in asphyctic brain are exclusively due to kynurenic acid formation. The possibility of a decreased efflux from the brain and/or increased influx from the circulation has to be considered. In the brain the levels of kynurenic acid are remarkably high during the prenatal period, and decrease dramatically around the time of birth [6, 7]. This phenomenon, which has been described in many species including rats, monkeys and sheep [6, 7, 17], led to the speculation that a rapid decline of cerebral kynurenic acid levels immediately following birth is indispensable to avoid postnatal blockade of NMDA receptor function, which is known to be required for essential curtailment of cell migration in brain development [18]. The effect on NMDA receptors is selective [18]. Therefore we may speculate that the marked endogenous elevation of kynurenic acid in the neonatal asphyctic brain could have also implications for potential pathological development. In contrast to the suggestion that kynurenic acid may also have a positive effect on hemodynamic and respiratory control [8], we have just recently found that kynurenic acid alters dosedependently the mitochondrial respiratory parameters by lowering the efficacy of mitochondrial ATP synthesis [19]. Although, that study was performed on adult rat heart mitochondria it seems conceivable that brain mitochondria may react similarly. Nevertheless, ATP levels are decreased in neonatal brain after asphyxia [20]. Beside the known neuroprotective capacities of kynurenic acid, highly elevated kynurenic acid values, as found following 20 minutes of asphyxia, might affect mitochondrial respiratory parameters, and subsequently contribute to a further impairment of cell function. This hypothetical consideration is based on the coincidence of the peak of kynurenic acid elevation and increasing mortality following asphyxia. An increase in mortality has been observed after a high dose of intraventricularly applied kynurenic acid [16], although the soporific and death inducing effect of kynurenic acid could be due to paralysis of NMDA receptor action. Another research group have found only a moderate increase of kynurenic acid in the cerebrum (without cerebellum), one hour after 15 minutes of asphyxia [7]. The explanation for this remarkable difference can be due to the shorter recovery period (10 minutes) from asphyxia used here and to the inclusion of the cerebellum. A prominent kynurenic acid formation has been shown to be induced in the cerebellum after kainic acid [21]. In contrast to the kynurenic acid changes, the other L-kynurenine metabolite, 3- hydroxykynurenine was only moderately altered during all asphyctic periods. 3-hydroxy-kynurenine is known to be neurotoxic [22], but it is an intermediate for quinolinic acid synthesis [4]. A moderate increase of 3-hydroxy-kynurenine after 20 minutes of asphyxia might be in line with a report showing a delayed increase of quinolinic acid levels after transient cerebral is-
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chemia in the gerbil [23], and perhaps associated to the delayed neuronal death observed with the present model [24]. This increase was not found in the prior mentioned study [7]. L-kynurenine, which serves as a substrate for both kynurenines was not affected significantly by asphyxia, although a tendency to a decrease could be seen. This minor effect is probably due to the rich presence of L-kynurenine in the prenatal brain tissue [6, 7]. The formation of kynurenic acid in the brain is catalyzed by kynurenine amino transferases I and II [25] and their activity during prenatal [7] and postnatal periods (up to the 7th day) is very low [7, 26], with a different ontogenetic pattern of alteration [7]. Presumably there are no kynurenine amino transferase activity changes during acute asphyxia, since no alteration of kynurenine amino transferase was reported in hippocampus or cerebellum 4 days after transient cerebral ischemia [23], although acidosis [20, 27] could affect enzyme activity. In this respect, no change of kynurenine amino transferase activity was found in the brain during the acute period of the kainic acid epilepsy model, although the kynurenic acid levels were remarkably elevated [21, and see Ref.]. The presence of a third kynurenine amino transferase in the brain (Baran, personal observation) may also have implications for kynurenic acid enhancement. Regarding to the enhancement of kynurenic acid levels in the asphyctic brain, accumulated data indicate that the increased of co-substrate (e. g. a-keto acids) availability may play a dominant role, since L-kynurenine is present at high concentrations [6, 7] and is not altered by asphyxia. Indeed, increased co-substrate levels due to a deficiency of a-keto-glutarate dehydrogenase complex activity in asphyxia has been reported [28]. In addition a substantial increase of kynurenic acid formation in the presence of pyruvate has been shown in aglycemic tissue slices from 7 days old rats [29]. There are also indications that a stimulation of dopamine receptor lowers significantly kynurenic acid formation in forebrain of 7 days old rats [29]. Furthermore, the same authors suggested that an early postnatal cerebral kynurenic acid synthesis is exceptionally modulated by dopaminergic mechanisms [29]. A reduction of dopamine levels after 20 minutes of perinatal asphyxia [30] may be related to a marked elevation of kynurenic acid levels in the asphyctic brain. Kynurenic acid once released from the cell is rapidly removed from the brain via a probenecid-sensitive mechanism, at least in adult animals [31]. However, information about mechanism(s) related to kynurenic acid transport within and outside the cell in the fetal brain of normal and asphyctic conditions is still lacking and needs to be completed. In this respect, it is of special interest to investigate kynurenic acid metabolism in sudden infant death syndrom. Acknowledgments Supported by SOLCO Pharma Austria and MH-M (Swedish Medical Research Council, number 8669; FONDECYT-Chile, number 1000626). References 1. Silverstein FS, Chen R, Johnston MV. The glutamate analogue quisqualic acid is neurotoxic in striatum and hippocampus of immature rat brain. Neuroscience Letters 1986;71:13–8. 2. Hagberg H, Andersson P, Kjellmer I, Thiringer K, Thordstein M. Extracellular overflow of glutamate, aspar-
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25. Guidetti P, Okuno E, Schwarcz R. Characterization of rat brain kynurenine aminotransferase I and II. Journal of Neuroscience Research 1997;50:457–5. 26. Baran H, Schwarcz R. Regional differences in the ontogenetic pattern of kynurenine aminotransferase in the rat brain. Developmental Brain Research 1993;74:283–6. 27. Engidawork E, Chen Y, Dell’Anna E, Goiny M, Lubec G, Ungerstedt U, Andersson K, Herrera-Marschitz M. Effect of perinatal asphyxia on systemic and intracerebral pH and glycolysis metabolism in the rat. Experimental Neurology 1997;145:390–6. 28. Dunckelmann RJ, Ebinger F, Schulze A, Wanders RJ, Rating D, Mayatepek E. 2-ketoglutarate dehydrogenase deficiency with intermittent 2-ketoglutaric aciduria. Neuropediatrics 2000;31:35–8. 29. Schwarcz R, Poeggeler B, Rassoulpour A, Ceresoli-Borroni G, Hodgkins PS. Regulation of kynurenic acid levels in the developing rat brain. Amino Acids 1998;14:243–9. 30. Chen Y, Engidawork E, Loidl F, Dell’Anna E, Goiny M, Lubec G, Andersson K, Herrera-Marschitz M. Shortand long-term effects of perinatal asphyxia on monoamine, amino acid and glycolysis product levels measured in the basal ganglia of the rat. Developmental Brain Research 1997; 104: 19–30. 31. Moroni F, Russi P, Lombardi G, Beni M, Carla V. Presence of kynurenic acid in the mammalian brain. Journal of Neurochemistry 1988;51:177–80.
Increased kynurenic acid in the brain after neonatal asphyxia H. Barana,*, B. Kepplingerb, M. Herrera-Marschitzc, K. Stolzea, G. Lubecd, H. Nohla a
Institute of Pharmacology and Toxicology, Veterinary Medical University Vienna, A-1210 Vienna, Austria b Department of Neurology, Diagnostic and Therapy Center LNK, A-3362 Mauer/Amstetten, Austria c Department of Physiology and Pharmacology, 17177 Stockholm, Sweden d Department of Pediatrics, University of Vienna, A-1090 Vienna, Austria Received 17 October 2000; accepted 8 February 2001
Abstract
In the brain, L-kynurenine is an intermediate for the formation of kynurenic acid, a metabolite with neuroprotective activities, and a substrate for the synthesis of 3-hydroxy-kynurenine, a metabolite with neurotoxic properties. In the present study, alterations of L-kynurenine, 3-hydroxy-kynurenine and kynurenic acid levels were examined in the brain of neonatal (10 minutes old) rats after 5, 10, 15 or 20 minutes of asphyxia, and in the brain of the corresponding caesarean-delivered controls, using sensitive high-performance liquid chromatographic methods. Among kynurenines we found a marked time-dependent increase of kynurenic acid levels, a moderately delayed increase of 3-hydroxy-kynurenine, and a trend for a decrease of L-kynurenine content. Thus, the brain reacted rapidly to the oxygen deficit by increasing kynurenic acid levels by 44 % already after 5 minutes of asphyxia, and the most prominent elevation of kynurenic acid (302 % of control) was found after 20 minutes of asphyxia - the critical time limit of survival. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Asphyxia; Excitatory amino acids; Kynurenic acid; Neuroprotection; Mitochondria, Lethality
Introduction Perinatal asphyxia is considered to lead to a variety of pathological conditions and may be linked to psychiatric and neurologic disorders later in life. Experimental data suggest a role of excitatory amino acids (EAA) in the genesis of ischemic damage in the immature brain * Corresponding author. Institute of Pharmacology and Toxicology, Veterinary University Vienna, A-1210 Vienna, Austria. E-mail address: [email protected] (H. Baran) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 1 5 -2
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[1]. It has been shown that the exogenous excitatory amino acids e.g. quisqualate and N-methyl-D-aspartate (NMDA) are potent excitotoxins in the immature brain [1]. Glutamate and aspartate levels are significantly elevated in the brain during hypoxic-ischemia of immature lambs [2] and rats [30]. Experimental animal studies have shown that the non-competitive NMDA receptor blocker MK-801 attenuates brain damage induced by hypoxic-ischemic insults [3], and kynurenic acid, a well known antagonist of EAA receptors with a high affinity to the glycine site of the NMDA receptor complex [4], has been used as neuroprotectant and anticonvulsant in various experimental conditions [4, 5]. L-kynurenine and kynurenic acid levels have been found to be development-dependent. Thus, L-kynurenine and kynurenic acid levels decline rapidly after birth [6, 7]. Since it has been shown in 7 days old neonatal rats that kynurenic acid offered a partial neuroprotection after hypoxic-ischemia [8], we questioned the possibility of kynurenine metabolism alteration in the asphyctic brain. In addition, we were interested on investigating another metabolite of L-kynurenine, 3-hydroxykynurenine, which declines after birth [7], but may display neuronal cytotoxicity [4]. Thus, the aim of present work was to study the endogenous levels of L-kynurenine, kynurenic acid and 3-hydroxy-kynurenine in the rat brain immediately after birth (10 minutes of age) following different degrees of perinatal asphyxia. A preliminary account of this work has been presented elsewhere [9]. Methods Materials L-kynurenine, kynurenic acid and 3-hydroxy-kynurenine were purchased from Sigma. All other chemicals used were also of the highest commercially available purity. Animal experiments Asphyxia was induced in pups delivered by a Caesarean section on pregnant SpragueDawley rats as described earlier [10]. In the present model, rats within the last day of gestation (as evaluated by protocols from the Animal Department), were anaesthetized with ether and hysterectomised. The uterus horns, still containing the fetuses were taken out, and placed into a water bath at 378C for various periods of time (5 to 20 minutes). Immediately after hysterectomy one or two pups were delivered to be considered caesarean-delivered (0 min asphyxia) controls, before the uterus horns with the remaining fetuses were placed into the water bath. Thus, control and asphyctic pups were obtained from the same mother, since each rat provided approximately 10–14 pups. After the corresponding asphyctic periods at 378C, the uterus horns were rapidly opened and the pups were removed and stimulated to breathe by cleaning the amniotic fluid and tactile stimulation of the oral region with pieces of medical wipes. The pups were intensively stimulated for approximately 5 minutes until a gasp was produced, or a decision was made that the pups were unable to recover. Further intermittent stimulation was performed on a heating pad to be sure that regular breathing was established, and thereafter killed by decapitation 10 minutes after the corresponding asphyctic periods. Control pups were decapitated 10 minutes after caesarean-delivery. The brain (cerebrum and cerebellum) immediately was removed and stored at 2808C pending further analysis. The
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animal studies were approved by the local Ethical Committee (Stockholm’s Norra Djurförsöksetiska Nämnd). Tissue preparation and neurochemical analysis Brains were homogenized by a Potter followed by a Polytron (Kinematics GmbH, Luzern, Switzerland) on an ice bath in 5 volumes (wt/vol) of 0.1 M HCl. The homogenates were centrifuged at 18,000 rpm for 15 min at 48C and one ml of the supernatant was applied to a Dowex 50 W cation exchange column pre-washed with 0.1 M HCl. Thereafter, the column was washed again with 1 ml 0.1 M HCl and 1 ml distilled water, and kynurenic acid was eluted with 2 ml distilled water [11]. L-kynurenine and 3-hydroxy-kynurenine were eluted with 2 ml of 1 M NH4OH [12]. The aliquots were frozen, lyophilized and then stored at 2308C until analysis. Prior to analysis the samples were re-suspended in 250 ml of distilled water for kynurenic acid, or in 250 ml of 0.1 M HCl for L-kynurenine and 3-hydroxy-kynurenine measurements, respectively. Kynurenic acid was measured by high performance liquid chromatography (HPLC) coupled to a fluorescence detection system, as previously reported [13]. The HPLC system used for analysis of kynurenic acid consisted of the following: a pump (Shimadzu, LC-6A), a fluorescence detector (Shimadzu, RF-535) set at an excitation wavelength of 340 nm, an emission wavelength of 398 nm, and a Shimadzu C-R5A Chromatopac Integrator. The mobile phase (isocratic system) consisted of 50 mM sodium acetate, 250 mM zinc acetate and 4 % acetonitrile, pH 6.2, and was pumped through a 10 cm 3 0.4 cm column (HR-80, C-18, particle size 3 mM, InChrom, Austria) at a flow rate of 1.0 ml/min, run at room temperature (238C). L-kynurenine and 3-hydroxy-kynurenine were measured by HPLC coupled with UV detector at 365 nm. The mobile phase contained 0.1 M ammonium acetate, 0.1 M acetic acid, and 2 % acetonitrile [14]. Protein determination Protein was measured according to the method of Bradford [15] using a commercially available kit (BIO-RAD) and bovine serum albumin as a standard. Statistics Kynurenine metabolite levels are per mg of protein. In addition we calculated the rate of kynurenic acid formation during the last 5 minutes for each period of asphyxia and expressed it as rate per minute. All data are given as means 6 S.E.M. For comparison of groups the Student t-test or Mann Whitney U-Test were applied. The level for statistical significance was p,0.05. Results In line with a prior study [10] we observed a progressive survival decline, in parallel with an increase of asphyxia time in perinatal pups. Until 15 minutes of asphyxia 100 % survival was observed. Survival declined to a 60 % after 20 minutes of asphyxia. We observed a moderate decrease of L-kynurenine content in the brain of asphyctic pups from 5 to 20 minutes of asphyxia (Table 1). Measurement of kynurenic acid revealed a significant and marked increase after all periods of asphyxia. Following 5 minutes of asphyxia the concentration of kynurenic acid was increased already by 44 % followed by 77 % after 10,
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Table 1 Kynurenine metabolites in the rat brain after different times of asphyxia Group (time of asphyxia)
L-kynurenine (pmol/mg protein)
Kynurenic acid (fmol/mg protein)
3-Hydroxykynurenine (fmol/mg protein)
13.09 6 0.69 (100 %) 11.75 6 0.68 (90 %) 11.63 6 1.17 (89 %) 12.22 6 0.73 (93 %) 11.31 6 1.08 (86 %)
335.86 17.1 (100 %) 482.29 653.1* (144 %) 593.6 6 59.0** (177 %) 726.0 6 73.3*** (216 %) 1013.7 6 62.6*** (302 %)
716.2 6 61.2 (100 %) 624.8 6 49.2 (87 %) 673.9 6 36.0 (94 %) 770.1 6 66.2 (108 %) 858.3 6 54.0* (120 %)
0 minutes (control) 5 minutes 10 minutes 15 minutes 20 minutes
Data represent a mean 6 S.E.M. of 10 control and 9–10 asphyxia animals. In parenthesis, % of control. Significances by using Mann Whitney U-Test: * p,0.05; ** p,0.01; *** p,0.01 vs control.
116 % after 15 and 202 % after 20 minutes, as compared to the controls. The highest rate of kynurenic acid formation during the last 5 minutes for each asphyxia period occurred after 20 minutes of asphyxia (Table 2). 3-Hydroxy-kynurenine declined moderately after 5 minutes of asphyxia, to be then normalized after 10, and moderately, but significant increased after 20 minutes of asphyxia.
Discussion Perinatal asphyxia is a major cause of death or neurological injury in newborn infants. In experimental animals it has been shown that the number of surviving pups declined significantly with an increase of the length of asphyxia [10]. In line with that study a 100 % of survival was observed up to 15 minutes of asphyxia, while only 60 % of the pups survived after 20 minutes of asphyxia. Asphyxia for longer than 21 minutes at 378C led to 100 % of mortality [10]. Among the investigated kynurenines e.g. L-kynurenine, kynurenic acid and 3-hydroxykynurenine, only kynurenic acid was markedly altered after asphyxia. The brain reacted rapidly to oxygen deficit (already after 5 minutes of asphyxia) by increasing kynurenic acid levels. The elevation of kynurenic acid levels was time-dependent. The rate of kynurenic acid elevaTable 2 Rate of kynurenic acid formation in the brain after different periods of asphyxia Rate of kynurenic acid formation 5 Minutes periods Kynurenic acid [fmol/mg protein/minute]
0–5 minutes
5–10 minutes
10–15 minutes
15–20 minutes
29.3 6 3.2
22.3 6 2.2
26.5 6 2.7
57.5 6 3.6***
Kynurenic acid formation rate was integrated for the last 5 minutes of each asphyxia period and calculated per minute. Data represent a mean 6 S.E.M. of 9–10 animals. Statistic significance was tested with the Student t-test: *** P , 0.001 versus the first 5 minutes-period.
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tion calculated per minute within 5 minute-periods was remarkably stable until 15 minutes of asphyxia, whereas after 20 minutes i.e. within the 4th 5 minute-period, the rate suddenly doubled. This indicates that mortality of asphyctic pups coincided with the highest rate of endogenous kynurenic acid formation. Interestingly, the evaluation of adult rats behaviour after intraventricular kynurenic acid application revealed dose-dependent changes, including stereotypy, ataxia, sleep and death, respectively [16]. On the other hand, intraperitoneally applied kynurenic acid offered a partial neuronal protection for hypoxic-ischemia in 7 days old neonatal rats, however the respiratory frequency and behaviour of the animals did not differ among treated and non-treated hypoxic-ischemia animals [8]. At present, there is no direct evidence that high kynurenic acid levels in asphyctic brain are exclusively due to kynurenic acid formation. The possibility of a decreased efflux from the brain and/or increased influx from the circulation has to be considered. In the brain the levels of kynurenic acid are remarkably high during the prenatal period, and decrease dramatically around the time of birth [6, 7]. This phenomenon, which has been described in many species including rats, monkeys and sheep [6, 7, 17], led to the speculation that a rapid decline of cerebral kynurenic acid levels immediately following birth is indispensable to avoid postnatal blockade of NMDA receptor function, which is known to be required for essential curtailment of cell migration in brain development [18]. The effect on NMDA receptors is selective [18]. Therefore we may speculate that the marked endogenous elevation of kynurenic acid in the neonatal asphyctic brain could have also implications for potential pathological development. In contrast to the suggestion that kynurenic acid may also have a positive effect on hemodynamic and respiratory control [8], we have just recently found that kynurenic acid alters dosedependently the mitochondrial respiratory parameters by lowering the efficacy of mitochondrial ATP synthesis [19]. Although, that study was performed on adult rat heart mitochondria it seems conceivable that brain mitochondria may react similarly. Nevertheless, ATP levels are decreased in neonatal brain after asphyxia [20]. Beside the known neuroprotective capacities of kynurenic acid, highly elevated kynurenic acid values, as found following 20 minutes of asphyxia, might affect mitochondrial respiratory parameters, and subsequently contribute to a further impairment of cell function. This hypothetical consideration is based on the coincidence of the peak of kynurenic acid elevation and increasing mortality following asphyxia. An increase in mortality has been observed after a high dose of intraventricularly applied kynurenic acid [16], although the soporific and death inducing effect of kynurenic acid could be due to paralysis of NMDA receptor action. Another research group have found only a moderate increase of kynurenic acid in the cerebrum (without cerebellum), one hour after 15 minutes of asphyxia [7]. The explanation for this remarkable difference can be due to the shorter recovery period (10 minutes) from asphyxia used here and to the inclusion of the cerebellum. A prominent kynurenic acid formation has been shown to be induced in the cerebellum after kainic acid [21]. In contrast to the kynurenic acid changes, the other L-kynurenine metabolite, 3- hydroxykynurenine was only moderately altered during all asphyctic periods. 3-hydroxy-kynurenine is known to be neurotoxic [22], but it is an intermediate for quinolinic acid synthesis [4]. A moderate increase of 3-hydroxy-kynurenine after 20 minutes of asphyxia might be in line with a report showing a delayed increase of quinolinic acid levels after transient cerebral is-
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chemia in the gerbil [23], and perhaps associated to the delayed neuronal death observed with the present model [24]. This increase was not found in the prior mentioned study [7]. L-kynurenine, which serves as a substrate for both kynurenines was not affected significantly by asphyxia, although a tendency to a decrease could be seen. This minor effect is probably due to the rich presence of L-kynurenine in the prenatal brain tissue [6, 7]. The formation of kynurenic acid in the brain is catalyzed by kynurenine amino transferases I and II [25] and their activity during prenatal [7] and postnatal periods (up to the 7th day) is very low [7, 26], with a different ontogenetic pattern of alteration [7]. Presumably there are no kynurenine amino transferase activity changes during acute asphyxia, since no alteration of kynurenine amino transferase was reported in hippocampus or cerebellum 4 days after transient cerebral ischemia [23], although acidosis [20, 27] could affect enzyme activity. In this respect, no change of kynurenine amino transferase activity was found in the brain during the acute period of the kainic acid epilepsy model, although the kynurenic acid levels were remarkably elevated [21, and see Ref.]. The presence of a third kynurenine amino transferase in the brain (Baran, personal observation) may also have implications for kynurenic acid enhancement. Regarding to the enhancement of kynurenic acid levels in the asphyctic brain, accumulated data indicate that the increased of co-substrate (e. g. a-keto acids) availability may play a dominant role, since L-kynurenine is present at high concentrations [6, 7] and is not altered by asphyxia. Indeed, increased co-substrate levels due to a deficiency of a-keto-glutarate dehydrogenase complex activity in asphyxia has been reported [28]. In addition a substantial increase of kynurenic acid formation in the presence of pyruvate has been shown in aglycemic tissue slices from 7 days old rats [29]. There are also indications that a stimulation of dopamine receptor lowers significantly kynurenic acid formation in forebrain of 7 days old rats [29]. Furthermore, the same authors suggested that an early postnatal cerebral kynurenic acid synthesis is exceptionally modulated by dopaminergic mechanisms [29]. A reduction of dopamine levels after 20 minutes of perinatal asphyxia [30] may be related to a marked elevation of kynurenic acid levels in the asphyctic brain. Kynurenic acid once released from the cell is rapidly removed from the brain via a probenecid-sensitive mechanism, at least in adult animals [31]. However, information about mechanism(s) related to kynurenic acid transport within and outside the cell in the fetal brain of normal and asphyctic conditions is still lacking and needs to be completed. In this respect, it is of special interest to investigate kynurenic acid metabolism in sudden infant death syndrom. Acknowledgments Supported by SOLCO Pharma Austria and MH-M (Swedish Medical Research Council, number 8669; FONDECYT-Chile, number 1000626). References 1. Silverstein FS, Chen R, Johnston MV. The glutamate analogue quisqualic acid is neurotoxic in striatum and hippocampus of immature rat brain. Neuroscience Letters 1986;71:13–8. 2. Hagberg H, Andersson P, Kjellmer I, Thiringer K, Thordstein M. Extracellular overflow of glutamate, aspar-
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