Excitatory amino acids and taurine levels in cerebrospinal fluid of hypoxic ischemic encephalopathy in newborn

Clinical Neurology and Neurosurgery 101 (1999) 171 – 174 www.elsevier.com/locate/clineuro

Excitatory amino acids and taurine levels in cerebrospinal fluid of hypoxic ischemic encephalopathy in newborn Kıvılcım Gu¨cu¨yener a,*, Yıldız Atalay b, Yusuf Ziya Aral c, Alev Hasanog˘lu d, Canan Tu¨rkyılmaz b, Gu¨rsel Biberog˘lu d a

Department of Pediatric Neurology, Gazi Uni6ersity Medical Faculty, Bukulm Sok. 9 /5, Ka6aklidere Ankara, Turkey b Department of Neonatology, Gazi Uni6ersity Medical Faculty, Bukulm Sok. 9 /5, Ka6aklidere Ankara, Turkey c Department of Pediatrics, Gazi Uni6ersity Medical Faculty, Bukulm Sok. 9 /5, Ka6aklidere Ankara, Turkey d Department of Pediatric Metabolism and Nutrition, Gazi Uni6ersity Medical Faculty, Bukulm Sok. 9 /5, Ka6aklidere Ankara, Turkey Received 25 November 1998; received in revised form 19 January 1999; accepted 3 May 1999

Abstract The recent studies indicating the transiently enhanced expression of excitatory amino acid receptors in hypoxia vulnerable brain regions and the elevated concentration of aspartate and glutamate in cerebrospinal fluid of asphyxiated newborns strongly suggest the role of excitatory amino acids in hypoxic ischemic brain damage in the developing human brain. In this study, we compared the concentrations of glutamate, aspartate, taurine and glycine in the cerebrospinal fluid of asphyxiated infants with values of a healthy control group. The concentrations of aspartate (5.829 3.36), glutamate (1.76 9 1.0) and taurine (9.329 9.1) were significantly elevated in cerebrospinal fluid of asphyxiated infants (P B0.05). When compared to the control group, the high levels of aspartate was correlated with the degrees of hypoxic-ischemic encephalopathy (HIE) and the varying outcome. The high levels of aspartate and glutamate in the asphyxiated patients adds further evidence to the role of excitotoxicity in hypoxic ischemic encephalopathy. The mental and motor development of the patients in asphyxiated group was followed for 3 years. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Excitatory amino acids; Taurine; Hypoxic ischemic encephalopathy; Newborn; Long term prognosis

1. Introduction Ischemic brain injury in the neonate is an important cause of neurologic disability in children [1,2]. The intrinsic excitatory amino acids (EAA) glutamate (GLU) and aspartate (ASP) and EAA agonists are capable of inducing an excitotoxic effect and were shown to be highly toxic when applied to the immature brain [3–5]. Furthermore, the neurons with high densities of EAA receptors are vulnerable to hypoxia ischemia and EAA antagonists reduce the degree of hypoxic brain damage when applied [6 – 8]. The most active phase of receptor development and the peak of

* Corresponding author. Tel.: +9-312-4185158; fax: + 9-3122150143.

EAA uptake sites occur in early postnatal life [6–9], and when a hypoxic ischemic insult occurs during this period the orderly progression of organization and synapse formation may be disturbed. The recent studies indicating the transiently enhanced expression of EAA receptors in asphyxia vulnerable brain region [10] and the elevated concentration of ASP and GLU in cerebrospinal fluid (CSF) of asphyxiated newborns strongly suggest the role of EAA in hypoxic ischemic brain damage in the developing human brain [11,12]. The literature contains very few data on EAAs in severe hypoxic encephalopathy of newborns in the clinical settings. Starting from that point of view in this study we compared the concentration of EAAs and the trend of an inihibitory amino acid taurine (TAU) in the asphyxiated groups. in the CSF of asphyxiated infants with that of a healthy control group. The asphyxiated group was followed up to 3 years of age.

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2. Patients and methods The study protocol was approved by the ethics committee of the University and a parental consent was asked for each infant. Twenty-two infants (ten term, 12 preterm) born between 30 and 39 weeks gestation were studied. All of them had their Apgar score of B5 at 5 min and cord pH B7.1, they were evaluated with respect to the grade of hypoxic-ischemic encephalopathy (HIE) [13] as: mild:hyperalertness, normal or weak suck reflex, normal muscular tone, no seizure. Moderate:weak or absent suck reflex, decreased level of consciousness, decreased muscular tone and frequent seizure. Severe:absent primitive reflexes, stupor or coma and usually without seizures. Infants with congenital infections, malformations or other diseases were excluded. Non-asphyxiated, neurologically healthy five preterm five term newborn infants (negative CSF cultures, Apgar score\ 8 at 1 min, lack of HIE and seizures) under clinical investigations of respiratory difficulties or infections served as controls. CSF was sampled 169 2 h after birth in the asphyxiated group and at 249 18 h in the controls respectively and immediately frozen at −70°C. After thawing the CSF samples were ultrafiltrated, and amino acids were determined using high performance liquid chromatography after precolumn devitalization [14]. All the concentrations of amino acids are expressed as means 9S.D. Mann – Whitney U test was used to evaluate differences.

3. Results The concentrations of ASP (5.829 3.36), GLU (1.76 9 1.0) and TAU (9.3299.1) were significantly elevated in CSF of asphyxiated infants (Table 1). The CSF concentration of GLU (2.6590.84) (PB 0.05), and TAU (21.65914.29) of the patients with severe HIE were significantly higher (PB 0.05) than that of the concentration of the patients with mild HIE (Table 2).

There was no significant difference between the amino acid concentrations of premature and term babies. The asphyxiated infants were followed up until the age of 3 years examined by a pediatric neurologist at 6, 9, 12, 24, 36 months. Disabilities, abnormalities in tones or reflexes, convulsions, blindness, hearing loss and microcephaly were classified as abnormal outcome. The CSF levels of aspartate were significantly higher in the patients with abnormal outcome when compared to that of the levels of the normals and the deceased patients (PB 0.05). However, the taurine levels were significantly elevated both in the patients with abnormal outcome and the ones who dies (P B 0.05) (Table 3).

4. Discussion A wide variety of acute and chronic neurologic disease may be mediated at least in part by excessive stimulation of glutamate receptors [15]. A bulk of evidence concerning the research in experimental animals have implicated the role for the EAA glutamate in the production of hypoxic-ischemic brain damage in the immature and adult brain, GLU is directly toxic to mature neurons in culture [16]. Neurons in hippocampal slices die when exposed to anoxia, but this detrimental effect can be avoided by magnesium which blocks glutamate receptors within the calcium (Ca2 + ) channel or by specific GLU antagonists [17,18]. Direct injection of GLU or its agonists into specific regions of brain in vivo produce neuronal injury identical to that seen after hypoxia ischemia. Finally, differentiation of the glutaminergic excitatory input into the hippocampus reduces the injury made by hypoxia-ischemia [19– 21]. Overstimulation of neuronal surface receptors by glutamate induces cell death, this GLU release from the axon terminal into the snaptic cleft occurs during hypoxia-ischemia [16] and the high levels in CSF could be the results of an extracellular over flow from the brain. The similar pattern of increased EAAs were found in

Table 1 Amino acid values in controls and asphyxiated infants Amino acids (mmol/l)

Glycine Aspartate Glutamate Taurine

Control group (n= 10)

Asphyxiated group (n =10)

Mean

Range

Sum of rank numbers

Mean

Range

Sum of rank numbers

1.8 2.2 0.8 4.4

2.5 1.6 0.9 2.7

141 78 93.5 99

2.7 5.8 1.8 10.6

6.8 11.9 3 38

387 450 434.5 429

* According to Mann–Whitney U test, the mean values are not significant. ** According to Mann–Whitney U test, the mean values are significant.

Conclusion

* ** ** **

K. Gu¨cu¨yener et al. / Clinical Neurology and Neurosurgery 101 (1999) 171–174

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Table 2 Amino acid values in mild and severe asphyxiated infants Amino acids (mmol/l)

Glycine Aspartate Glutamate Taurine

Mild asphyxiated (n =10)

Severe asphyxiated (n = 6)

Mean

Range

Sum of rank numbers

Mean

Range

Sum of rank numbers

2 4 1.4 5.9

2.2 4.9 3 5.7

74 67 64.5 56

2.5 6.8 2.7 21.7

3.1 10.5 2.2 32.9

62 69 71.5 80

Conclusion

* * ** **

* According to Mann–Whitney U test, the mean values are not significant. ** According to Mann–Whitney U test, the mean values are significant.

the microdialysis of the extracellular fluid in cerebral cortex and stiatum of hypoxic ischemic fetal lambs and neonatal rats [22,23]. Increased levels of EAAs in the CSF have been found previously in children with Rett Syndrome, infantile spasms and in hypoglycemic neonates [24 – 26]. The high concentration of GLU and ASP was shown to be present in the CSF of asphyxiated neonates and the concentration of EAAs in the CSF correlated well with the severity of hypoxia-ischemia [11,12]. In our study, in accordance with the previous results asphyxiated neonates had considerably higher CSF concentration of GLU and ASP. Also the increased CSF concentration of GLU well correlated with the degree of hypoxic-ischemic onset as being the highest in the severe hypoxia group. When the augmented release of EAAs initiate cellular damage; parallel to excitotoxicity some neurotransmitters may be involved in autoprotective cellular mechanisms and counteract the excitotoxicity of EAAs. Neurotransmitters like adenosine [27] and TAU [28] have to be recalled here. The extracellular concentrations of TAU has been shown to increase after hypoxia and ischemia in the immature brain parallel to the increment in extracellular glutamate and aspartate concentrations [29]. Biochemically TAU is an accumulation product of oxidation of cystine, cysteine, cysteamine and other sulphur-containing compounds considered to be natural scavengers of oxygen derived free radicals. It is also known as a Ca2 + transport modulator attenuating

Table 3 CSF amino acid values in varying outcome of asphyxiated infants Amino acids (mmol/l)

Abnormal (n =6) (mean9 S.D.)

Dead (n=4) (mean 9 S.D.)

Normal (n= 12) (mean9 S.D.)

Aspartate Glutamate Taurine

9.8 9 3.37* 2.02 9 0.99 12.58 914.24*

5.33 9 2.36 1.79 0.78 14.969 11.03*

3.989 1.52 1.65 9 1.1 5.89 2.04

* PB0.05.

Ca2 + influx and overload in brain tissue upon hypoxia [28,30]. In our hypoxic group of patients the high levels of CSF TAU differs from the previous two studies as discussed above could clearly indicate the protective function of this amino acid in hypoxia. The age related difference in the sensitivity of the brain to excitatory neurotransimitter toxicity arises from developmental alterations in the density and distribution of glutamate receptor subtypes [31]. It also depends on GLU binding to its receptors and the transmembrane biochemical events triggered by the receptor itself. Although this age related variations exist there was no difference between the values of term or preterm infants CSF EAAs. The high concentration of CSF GLU, ASP, TAU found in hypoxic infants and the correlation of the increased level of GLU with the severity of hypoxia and the final neurological outcome of he infants when classified due to their disabilities showed the highest correlation with the CSF aspartate and taurine levels are a further clinical evidences of the susceptibility of the immature brain to hypoxia and its long term effects. We have a minute number of patients on follow up. The fact of CSF aspartate levels being higher in the dead patients than that of the control group although not as the sole determinant this observation could indicate along with the others aspartate as one of the biochemical markers of HIE with regard to prognosis. Regarding the therapeutic intervention that will be put into action in near future earliest identification of those infants at highest risk for permenant brain damage is of utmost importance. Equally important is the understanding of the need for more clinical studies aimed to uncover the early and late effects of the cascading biochemical events of HIE. The direct overstimulation of GLU receptors by EAAs, variations in GLU receptor sensitivity, variations in number of GLU receptors and abnormalities or absence of appropriate uptake or degradative systems are the early finger prints of hypoxia with regard to long term prognosis should all be investigated thoroughly in all newborns with HIE.

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