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Relationship between glutamate, GOT and GPT levels in maternal and fetal blood: A potential mechanism for fetal neuroprotection
Early Human Development 88 (2012) 773–778
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Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Relationship between glutamate, GOT and GPT levels in maternal and fetal blood: A potential mechanism for fetal neuroprotection Alexander Zlotnik a,⁎, 1, Svetlana Tsesis a, 1, Benjamin Fredrick Gruenbaum a, Sharon Ohayon a, Shaun Evan Gruenbaum b, Matthew Boyko a, Eyal Sheiner c, Evgeny Brotfain a, Yoram Shapira a, Vivian Itzhak Teichberg d a
Department of Anesthesiology and Critical Care, Soroka Medical Center, Ben Gurion University of the Negev, Faculty of Health Sciences, Beer Sheva, Israel Department of Anesthesiology, Yale University School of Medicine, New Haven, CT, USA c Department of Obstetrics and Gynecology, Soroka Medical Center, Ben Gurion University of the Negev, Faculty of Health Sciences, Beer Sheva, Israel d Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel b
a r t i c l e
i n f o
Article history: Received 28 January 2012 Received in revised form 11 April 2012 Accepted 4 May 2012 Keywords: Fetal and maternal glutamate Glutamate-pyruvate transaminase (GPT) Glutamate-oxaloacetate transaminase (GOT) Neurotoxicity
a b s t r a c t Background: Excess glutamate in the brain is thought to be implicated in the pathophysiology of fetal anoxic brain injury, yet little is known about the mechanisms by which glutamate is regulated in the fetal brain. This study examines whether there are differences between maternal and fetal glutamate concentrations, and whether a correlation between them exists. Methods: 10 ml of venous blood was extracted from 87 full-term (>37 weeks gestation) pregnant women in active labor. Immediately after delivery of the neonate, 10 ml of blood from the umbilical artery and vein was extracted. Samples were analyzed for levels of glutamate, glutamate‐oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT). Results: Fetal blood glutamate concentrations in both the umbilical artery and vein were found to be significantly higher than maternal blood (p b 0.001). Similarly, fetal serum GOT levels in the umbilical artery and vein were found to be significantly higher than maternal GOT levels (p b 0.001). The difference in GPT levels between maternal and fetal serum was not statistically significant. There was no difference in fetal glutamate, GOT or GPT between the umbilical artery and vein. There was an association observed between glutamate levels in maternal blood and glutamate levels in both venous (R = 0.32, p b 0.01) and arterial (R = 0.33, p b 0.05) fetal blood. Conclusions: This study demonstrated that higher baseline concentrations of blood glutamate are present in fetal blood compared with maternal blood, and this was associated with elevated GOT, but not GPT levels. An association was observed between maternal and fetal blood glutamate levels. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Birth asphyxia is a leading cause of neurological pathology and death in newborns [1]. Elevated L-glutamate (glutamate) concentrations in the brain's extracellular fluids (ECF) are associated with a worse neurological outcome after fetal asphyxia. Following a hypoxic event, the activation of glutamatergic receptors initiates a cascade of events that results in cellular necrosis and apoptosis [1–3]. There have been great efforts in recent years to discover potential methods of preventing the sequelae that follows excess glutamate in the brain, however no such treatment exists as of yet.
⁎ Corresponding author at: Department of Anesthesiology and Critical Care, Soroka Medical Center, POB 151, Beer Sheva, 84105, Israel. Tel.: + 972 8 6400262; fax: + 972 8 6403795. E-mail address: [email protected] (A. Zlotnik). 1 Equal contribution. 0378-3782/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2012.05.001
Following brain injury, glutamate transporters on brain capillary endothelial cells play an important role in the redistribution of glutamate from the brain to the blood [4,5]. In rat studies, the brainto-blood efflux of glutamate was accelerated by increasing the concentration gradient between the CSF and plasma [6]. Decreasing blood glutamate concentrations increases the brain-to-blood efflux and improves neurological outcomes after TBI in rats [7–9]. This was achieved by activating the blood resident enzymes glutamate-pyruvate transaminase (GPT) and glutamate-oxaloacetate transaminase (GOT), which in the presence of their co-substrates pyruvate and oxaloacetate, convert glutamate into 2-ketoglutarate [6]. Similarly, by artificially increasing plasma glutamate concentrations, glutamate concentrations in the brain's extracellular fluids were increased [5]. Campos et al. demonstrated that lower blood glutamate levels and higher levels of GOT were associated with a better neurological outcome in patients after ischemic stroke [10–12]. Potential factors that increase blood glutamate levels subsequently increase brain glutamate
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levels, and may result in a worse neurological outcome after global ischemia, brain injury, or stroke. Furthermore, potential factors that reduce blood glutamate concentrations may lead to a decrease in brain glutamate levels, thereby providing neuroprotection. It is known that the placenta has an abundance of glutamate transporters, and is capable of removing of excess of glutamate by active transport from the fetal to maternal blood [13,14]. Amino acid concentrations in the fetus are higher compared with concentrations in the mother [14,15]. The glycine–glutamate turnover cycle between the fetal circulation and placenta has been well described. Glycine is transported from the mother into the fetal circulation by active transport, and is converted to glutamate predominantly in the fetal liver. When excess glutamate is present in the fetal circulation, the glutamate is transported to the placenta to prevent glutamateinduced neurotoxicity. The placenta is responsible for the metabolism of as much as 80% of fetal glutamate [13,14]. Based on our prior studies with blood glutamate scavengers GOT, GPT, oxaloacetate, and pyruvate, we theorize that fetal blood glutamate concentrations may potentially be reduced via a reduction of maternal blood glutamate levels. If this is indeed possible, this decrease in fetal blood glutamate concentrations would be expected to result in the fast transport of excess glutamate from the fetal brain to the blood, thereby providing neuroprotection. This is especially important because this would provide the potential to begin early treatment in utero, with the continuation of treatment after delivery. The purpose of this study was to investigate the relationship between concentrations of glutamate in the maternal blood and fetal blood (umbilical artery and vein), to determine the concentrations of GOT and GPT and their correlation with glutamate levels, and to determine the relationship between fetal glutamate levels and fetal outcomes. 2. Materials and methods This experiment has been conducted according to the recommendations set by the Helsinki Committee and was approved by the Ethics Committee of Ben Gurion University of the Negev, Beer Sheva, Israel. Each participant signed an informed consent before the beginning of the experiment. 2.1. Studied population A total of 87 pregnant females between the ages of 18 and 40 participated in the study. For the purpose of determining a correlation between blood glutamate, GOT and GPT concentrations in different conditions, mothers were included in the study irrespective to any coexisting pathology, method of delivery (cesarean section or vaginal), or type of anesthesia or analgesia in cases of cesarean section or vaginal delivery, respectively. 2.2. Experimental design Enrolled mothers in the study were followed up during their labor. Immediately after delivery and the clamping of the umbilical cord, blood samples were extracted simultaneously from the maternal peripheral vein, umbilical artery and umbilical vein into pre-marked syringes. Volumes of each blood sample were measured at approximately 5 ml. Immediately after blood samples collection, 0.2 ml of blood was removed from each blood sample for the determination of glutamate levels in whole blood, as described below. The remainder of each blood sample was used to measure serum GOT and GPT concentrations, as described below. The following data was collected from medical records: age of parturients, age of the pregnancy, symptoms of fetal distress (listed below), sex of newborns, co-existing conditions (both maternal and fetal) and method of delivery (vaginal or cesarean section).
Fetal distress was considered when at least one of the following symptoms was registered during labor and immediately after delivery: fetal bradycardia (defined as fetal heart rate by monitor b 100 BPM), fetal tachycardia (defined as fetal heart rate by monitor > 160 BPM), late decelerations, variable decelerations, meconial, infectious or bloody amniotic fluids, Apgar score ≤ 7 in the first minute, necessity of newborn intubation for oxygenation maintenance, or umbilical artery blood pH on delivery ≤ 7.2. 2.3. GOT and GOT determination in serum Serum GOT and GPT levels were determined in the biochemical laboratory of Soroka Medical Center via a fluorescent method, based on the conversion of glutamate into alanine and aspartate in the presence of GOT and GPT respectively (Olympus AU 2700). 2.4. Glutamate determination in whole blood Whole blood (200 μl aliquot) was deproteinized by adding an equal volume of ice-cold 1 M perchloric acid (PCA) and then centrifuging at 10,000 ×g for 10 min at 4 °C. The pellet was discarded and 200 μl supernatant collected, adjusted to pH 7.2 with 50 μl 2 M K2CO3, repeatedly centrifuged at 10,000 ×g for 4 min at 4 °C and stored at −80 °C for later analysis (at a REVCO freezer). Glutamate concentrations were measured using the fluorometric method of Graham and Aprison [16]. A 20 μl aliquot from the PCA supernatant was added to 480 μl of a 0.3 M glycine, 0.25 M hydrazine hydrate buffer adjusted to pH 8.6 with 1 N H2SO4 and containing 15 U of glutamate dehydrogenase in 0.2 mM NAD. After incubation for 30–45 min at room temperature, the fluorescence was measured at 460 nm with excitation at 350 nm. A glutamate standard curve was established with concentrations ranging from 0 to 6 μM. All determinations were done at least in duplicates. 2.5. Statistics Levels of glutamate, GOT and GPT were compared between maternal, umbilical artery and umbilical vein samples using repeated measures analysis of variance with Bonferroni post‐hoc testing. Pearson's correlation coefficient was used to analyze the relationship between concentrations of glutamate, GOT and GPT in maternal, umbilical artery and umbilical vein blood. Statistical analysis was performed by using the SPSS 18.0 software package (SPSS, Inc, Chicago, IL). Differences in data were considered significant when p b 0.05 and highly significant when p b 0.01. Data are presented as average ± SEM. 3. Results 3.1. Subjects A total of 87 pregnant females between the ages of 18 and 40 participated in the study. The average maternal age was 26 ± 9 years. The average age of pregnancy was 38 ± 3 weeks. Of the 87 delivered newborns, 41 were females and remaining 46 were males. Of the 87 participants, we were unable to collect a blood sample from the umbilical artery in 8 women and were unable to collect a blood sample from the umbilical vein in 3 women. This was due to small volumes of blood in the artery or vein respectively or due to early clot formation. Fifty-five participants underwent a vaginal delivery. The remaining 32 delivered via cesarean section, of which 23 were anesthetized via general anesthesia and 9 were anesthetized with spiral or epidural anesthesia. General anesthesia included induction via preoxygenation, 2–3 mg/kg of propofol, and 1.5 mg/kg succinylcholine, with maintenance via isoflurane 1 MAC, N2O/O2 50:50, 1–2 μg/kg of fentanyl and either atracurium or rocuronium was used for muscle relaxation.
A. Zlotnik et al. / Early Human Development 88 (2012) 773–778
3.2. Blood glutamate concentrations Fetal blood glutamate concentrations, in both the umbilical artery and vein, were nearly twice as high as maternal blood glutamate concentrations (p b 0.001, Fig. 1). There were no significant differences between glutamate concentrations in the umbilical artery and umbilical vein. There was an association observed between glutamate levels in maternal blood and glutamate levels in both venous (R = 0.32, p b 0.01) and arterial fetal blood (R = 0.33, p b 0.05). There was a significant positive Pearson's correlation found between blood glutamate concentrations in arterial and venous fetal blood (R = 0.85, p b 0.001) (Fig. 2). 3.3. Serum GOT and GPT levels Measured serum GOT and GPT levels are presented in Table 1. Maternal serum GOT levels were found to be significantly lower than fetal serum GOT levels in the umbilical artery and vein (pb 0.001). There were no significant difference between GOT concentrations in umbilical artery and umbilical vein. On the contrary, GPT levels did not differ significantly between maternal serum, umbilical artery and umbilical vein. There was no significant correlation found between GOT or GPT levels in maternal and fetal serum. However, there was a strong correlation found between levels of GOT in arterial and venous fetal serum (R = 0.72, p b 0.001). 3.4. Correlation between symptoms of fetal distress and fetal blood glutamate levels There were 21 cases where the fetus or newborn presented with at least one of the symptoms of fetal distress listed above. Of them, meconial amniotic fluid was seen in 6 cases, a low Apgar score was observed in 5 cases, and a low pH was measured in 4 cases. Severe distress, based on an Apgar score of ≤7 in the first minute with concurrent umbilical artery blood pH on delivery of ≤7.2, was seen in only 4 cases. Five babies were diagnosed with intrauterine growth retardation at birth. There was no difference in glutamate levels found between those with or without fetal distress in either the umbilical artery or umbilical vein.
Fig. 1. Differences in blood glutamate concentrations. Fetal blood glutamate concentrations were found to be significantly higher than maternal blood levels in both the umbilical artery and vein (p b 0.001 *). There were no significant differences between the glutamate concentrations in the fetal umbilical artery and umbilical vein. Data are presented as means ± SEM.
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3.5. Correlation between gender of newborns and blood glutamate, GOT and GPT levels Forty-one of the 87 delivered newborns were female, and the remaining 46 were male. There were no statistically significant differences in blood glutamate levels between female and male newborns in arterial umbilical blood or venous umbilical blood. There were no significant differences in serum GOT levels between female and male newborns. There were no significant differences in serum GPT levels between female and male newborns. 4. Discussion In this study we demonstrated that glutamate concentrations in whole blood were significantly higher in both the umbilical arterial and venous blood compared with maternal blood. GOT levels were significantly higher in the umbilical arterial and venous serum compared with maternal serum, while GPT levels were not significantly different. The concentration of glutamate in fetal blood correlated with the glutamate concentration in maternal blood. There was no difference in fetal glutamate, GOT or GPT between the umbilical artery and vein. It was previously shown that the concentrations of many amino acids in fetal plasma exceed the concentrations in maternal plasma [15]. The high glutamate concentrations in fetal blood relative to maternal blood, as was demonstrated in this study, may not be due to a limited ability to clear glutamate. Rather, these higher concentrations likely reflect an important role for normal brain development. Excitatory amino acids were shown to be crucial in the development of brain plasticity in animals and humans, and the expression of glutamate receptors is enhanced in the developing animal and human brain. It has further been shown that plasma glutamate concentrations are maximally elevated in newborns, rapidly decrease during the first months of life, and then slowly decreases over the following several years [17,18]. As such, NMDA receptor blockade significantly influences the development of cortical plasticity [19]. Although significantly elevated glutamate levels after brain insults are implicated in secondary brain injury, NMDA receptor antagonists failed to provide neuroprotection in human studies with both TBI and stroke [20]. One possible explanation for this may be that glutamate, at normal concentrations in the brain's fluids, is critical for preserving normal neuronal function by activating glutamate receptor signaling (via both ionotropic and metabotropic receptors) and maintaining neuronal integrity. NMDA receptor antagonists interfere with both the negative and positive effects of this signaling [21,22]. Therefore, glutamate levels in both the brain's ECF and the blood require tight regulation to balance its positive and neurotoxic effects. It is known that GOT, an enzyme present in the blood, converts glutamate to its non-toxic metabolite 2-ketoglutarate. In this study, we observed that GOT levels in fetal serum were higher than maternal GOT levels. As expected from the Michaelis–Menten enzyme rate equation, higher levels of GOT are necessary to convert the increased amount of glutamate in fetal blood into 2-ketoglutarate. Our finding that the elevated concentration of GOT in fetal plasma directly correlates with the rise in glutamate suggests that the elimination of glutamate does not rely solely on the placenta. Rather, this elevation of GOT is likely necessary to maintain the adequate balance between the positive and negative effects of glutamate on the developing brain [19]. Hays and colleagues showed that dietary glutamate is almost entirely removed from the bloodstream via first pass metabolism through the splanchnic bed in premature infants [23]. This is not surprising considering the very high concentrations of GOT in newborns' serum, supporting the idea that the elevated GOT levels have high glutamate-converting capacity in the context of elevated blood glutamate levels. Conversely, GPT levels were not elevated in fetal serum, suggesting that the GPT-dependent mechanism of glutamate
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Fig. 2. The correlations between blood glutamate and GOT concentrations. The correlation between glutamate levels in maternal blood and glutamate levels in arterial fetal blood was R = 0.33 (p b 0.05; panel A). The correlation between glutamate levels in maternal blood and glutamate levels in venous fetal blood was R = 0.32 (p b 0.01; panel B).The correlation between blood glutamate concentrations in arterial and venous fetal blood was R = 0.85 (p b 0.001; panel C). The correlation between GOT in arterial and venous fetal serum was R = 0.72 (p b 0.001; panel D).
metabolism plays a less significant role than GOT. The limited significance of GPT compared with GPT reflects previous observations in adult humans after ischemic stroke [10–12] and in rats after TBI [24]. The literature regarding glutamate concentrations in fetal or newborns’ plasma compared to maternal concentrations is inconsistent. Cetin and colleagues reported that glutamate concentrations in the umbilical artery is 1.5 times lower than in the maternal plasma, while glutamate concentrations in the umbilical vein were two-fold lower than in the umbilical artery [14]. However, the authors employed Table 1 Measured serum GOT and GPT concentrations. Fetal serum GOT concentrations were found to be significantly higher than maternal serum levels in both the umbilical artery and vein (p b 0.001 *). The difference in GPT levels between maternal and fetal blood was not statistically significant. There was no difference in fetal GOT or GPT between the umbilical artery and vein. Data are presented as means ± SEM. Parameter
Maternal blood
Umbilical artery
Umbilical vein
Number of samples analyzed GOT (U/L) GPT (U/L)
87 19 ± 1 11 ± 1
79 39* ± 2 13 ± 1
84 38* ± 2 13 ± 1
a much smaller sample size than that of our study, and the discrepancy may be due to differences in the study design. Furthermore, while their study measured glutamate levels in maternal and fetal plasma, our findings reflect that of whole blood. Considering that the concentration of glutamate in erythrocytes is 4 to 5 times higher than in plasma [25], and that hemoglobin and hematocrit concentrations in the fetus and newborn are almost two times higher than in pregnant women, fetal whole blood has a much higher concentration of glutamate compared with maternal whole blood. Moreover, two other studies demonstrated that the concentration of glutamate in the plasma during one's lifetime is maximal in newborns, rapidly decreases during the first months of life, and then slowly decreases over the following several years [17,18]. If this is the case, it would not be possible for fetal or newborn blood glutamate levels to be lower than maternal levels. We demonstrated an association between maternal and fetal blood glutamate levels. This is an especially important finding because it suggests that under fetal asphyxia conditions, one may theoretically reduce fetal blood glutamate concentrations by decreasing maternal blood glutamate levels. However, causality in the observed association between
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maternal and fetal glutamate, GOT and GPT levels cannot yet be presumed and additional experimental studies are still necessary to test this hypothesis. Yet, these findings may initiate future studies to explore the possibility of new treatment strategies directed at decreasing glutamate concentration in the fetal brain, promoting neuroprotection without affecting the vitally important functions of glutamate in the developing brain. There were no differences observed in fetal glutamate, GOT or GPT between the umbilical artery and vein. Glutamate is an amino acid that is intensively metabolized and synthesized in all compartments of the body: blood, peripheral tissues and placenta. A free shift of unchanged glutamate exists between these different compartments. In adults, for example, under normal conditions, blood glutamate levels are in a steady state regulated by a net export from the liver and a net utilization by the skeletal muscles and gut [26]. Therefore, considering this very complex mechanism of glutamate's metabolism with the concurrent synthesis and distribution in different tissues, one would assume different glutamate concentrations between venous and arterial blood in the fetus. Therefore, this observation was not as expected and is an important finding. We found no differences in blood glutamate levels between newborns with symptoms of fetal distress and those without symptoms of fetal distress. This finding was not surprising, for if fetal distress and asphyxia affects fetal blood glutamate levels, it is reasonable that this process would take at least several hours to develop. Today, it is common in obstetric practice to monitor the fetal condition during labor and respond immediately if symptoms of fetal distress appear. Usually, within a few minutes of the development of symptoms of fetal distress, the fetus is delivered via cesarean section or vaginally with a vacuum. However, in this study, there were a limited number of cases in which fetal distress was present precluding us from making any final conclusions regarding this issue. We previously showed that the female sex hormones estrogen and progesterone were inversely correlated with blood glutamate levels [27,28]. As such, in human studies blood glutamate and GOT levels were shown to be much lower in females than in males [27,28]. For this reason, we examined whether gender differences in blood glutamate levels exist in newborns. We did not find any significant differences in blood glutamate, GOT and GPT levels between female and male newborns. This may be explained by an equal concentration of female sex hormones in the male and female fetus and newborn due to an immaturity of their gonadal glands [29]. Considering that the placenta has a well-developed system of transporters for glutamate elimination, it seems plausible that one may create a favorable chain of glutamate concentration gradients that would ultimately lead to the elimination of excess glutamate from the fetal brain. This chain would start with the reduction of glutamate concentrations in the maternal blood compartment, thereby creating a favorable glutamate gradient between the maternal and fetal blood. This would lead to a placenta-mediated efflux of glutamate from the fetal to maternal blood. Decreases in fetal blood glutamate concentrations should then facilitate the efflux of excess of glutamate from the fetal brain's ECF compartment, thereby preventing its neurotoxic effects. The results of this study may offer new therapeutic insights into future treatment modalities of fetal blood glutamate reduction in utero by way of reducing maternal glutamate levels. In newborns, asphyxia has been shown to be associated with an elevated ECF glutamate concentration that slowly decreases toward baseline levels by 72 h [1]. Considering that fetal distress may be diagnosed early during labor via fetal monitoring, prompt initiation of the treatment may improve neurological outcome in newborns. While the efficacy of glutamate-reducing therapeutic options is not yet clear, potentially such treatment could be initiated “in utero” and continued after delivery. Additional experiments in both clinical and experimental settings are still warranted to investigate potential treatment strategies and their therapeutic window.
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In conclusion, we demonstrated that higher baseline concentrations of blood glutamate are present in fetal blood compared with maternal blood, and this was associated with an increase in GOT, but not GPT. An association was observed between maternal and fetal blood glutamate levels. These findings are important in investigating the possibility of new therapeutic strategies for fetal asphyxia conditions, via decreasing fetal blood glutamate concentrations in utero by reducing maternal glutamate levels.
Conflict of interest statement The authors state that no competing financial or other conflicts of interests exist.
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[23] Hays SP, Ordonez JM, Burrin DG, Sunehag AL. Dietary glutamate is almost entirely removed in its first pass through the splanchnic bed in premature infants. Pediatr Res 2007;62:353–6. [24] Zlotnik A, Gurevich B, Cherniavsky E, Tkachov S, Matuzani-Ruban A, Leon A, et al. The contribution of the blood glutamate scavenging activity of pyruvate to its neuroprotective properties in a rat model of closed head injury. Neurochem Res 2008;33:1044–50. [25] Fukuda K, Nishi Y, Usui T. Free amino acid concentrations in plasma, erythrocytes, granulocytes, and lymphocytes in umbilical cord blood, children, and adults. J Pediatr Gastroenterol Nutr 1984;3:432–9. [26] Klin Y, Zlotnik A, Boyko M, Ohayon S, Shapira Y, Teichberg VI. Distribution of radiolabeled l-glutamate and d-aspartate from blood into peripheral tissues in
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Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Relationship between glutamate, GOT and GPT levels in maternal and fetal blood: A potential mechanism for fetal neuroprotection Alexander Zlotnik a,⁎, 1, Svetlana Tsesis a, 1, Benjamin Fredrick Gruenbaum a, Sharon Ohayon a, Shaun Evan Gruenbaum b, Matthew Boyko a, Eyal Sheiner c, Evgeny Brotfain a, Yoram Shapira a, Vivian Itzhak Teichberg d a
Department of Anesthesiology and Critical Care, Soroka Medical Center, Ben Gurion University of the Negev, Faculty of Health Sciences, Beer Sheva, Israel Department of Anesthesiology, Yale University School of Medicine, New Haven, CT, USA c Department of Obstetrics and Gynecology, Soroka Medical Center, Ben Gurion University of the Negev, Faculty of Health Sciences, Beer Sheva, Israel d Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel b
a r t i c l e
i n f o
Article history: Received 28 January 2012 Received in revised form 11 April 2012 Accepted 4 May 2012 Keywords: Fetal and maternal glutamate Glutamate-pyruvate transaminase (GPT) Glutamate-oxaloacetate transaminase (GOT) Neurotoxicity
a b s t r a c t Background: Excess glutamate in the brain is thought to be implicated in the pathophysiology of fetal anoxic brain injury, yet little is known about the mechanisms by which glutamate is regulated in the fetal brain. This study examines whether there are differences between maternal and fetal glutamate concentrations, and whether a correlation between them exists. Methods: 10 ml of venous blood was extracted from 87 full-term (>37 weeks gestation) pregnant women in active labor. Immediately after delivery of the neonate, 10 ml of blood from the umbilical artery and vein was extracted. Samples were analyzed for levels of glutamate, glutamate‐oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT). Results: Fetal blood glutamate concentrations in both the umbilical artery and vein were found to be significantly higher than maternal blood (p b 0.001). Similarly, fetal serum GOT levels in the umbilical artery and vein were found to be significantly higher than maternal GOT levels (p b 0.001). The difference in GPT levels between maternal and fetal serum was not statistically significant. There was no difference in fetal glutamate, GOT or GPT between the umbilical artery and vein. There was an association observed between glutamate levels in maternal blood and glutamate levels in both venous (R = 0.32, p b 0.01) and arterial (R = 0.33, p b 0.05) fetal blood. Conclusions: This study demonstrated that higher baseline concentrations of blood glutamate are present in fetal blood compared with maternal blood, and this was associated with elevated GOT, but not GPT levels. An association was observed between maternal and fetal blood glutamate levels. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Birth asphyxia is a leading cause of neurological pathology and death in newborns [1]. Elevated L-glutamate (glutamate) concentrations in the brain's extracellular fluids (ECF) are associated with a worse neurological outcome after fetal asphyxia. Following a hypoxic event, the activation of glutamatergic receptors initiates a cascade of events that results in cellular necrosis and apoptosis [1–3]. There have been great efforts in recent years to discover potential methods of preventing the sequelae that follows excess glutamate in the brain, however no such treatment exists as of yet.
⁎ Corresponding author at: Department of Anesthesiology and Critical Care, Soroka Medical Center, POB 151, Beer Sheva, 84105, Israel. Tel.: + 972 8 6400262; fax: + 972 8 6403795. E-mail address: [email protected] (A. Zlotnik). 1 Equal contribution. 0378-3782/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2012.05.001
Following brain injury, glutamate transporters on brain capillary endothelial cells play an important role in the redistribution of glutamate from the brain to the blood [4,5]. In rat studies, the brainto-blood efflux of glutamate was accelerated by increasing the concentration gradient between the CSF and plasma [6]. Decreasing blood glutamate concentrations increases the brain-to-blood efflux and improves neurological outcomes after TBI in rats [7–9]. This was achieved by activating the blood resident enzymes glutamate-pyruvate transaminase (GPT) and glutamate-oxaloacetate transaminase (GOT), which in the presence of their co-substrates pyruvate and oxaloacetate, convert glutamate into 2-ketoglutarate [6]. Similarly, by artificially increasing plasma glutamate concentrations, glutamate concentrations in the brain's extracellular fluids were increased [5]. Campos et al. demonstrated that lower blood glutamate levels and higher levels of GOT were associated with a better neurological outcome in patients after ischemic stroke [10–12]. Potential factors that increase blood glutamate levels subsequently increase brain glutamate
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levels, and may result in a worse neurological outcome after global ischemia, brain injury, or stroke. Furthermore, potential factors that reduce blood glutamate concentrations may lead to a decrease in brain glutamate levels, thereby providing neuroprotection. It is known that the placenta has an abundance of glutamate transporters, and is capable of removing of excess of glutamate by active transport from the fetal to maternal blood [13,14]. Amino acid concentrations in the fetus are higher compared with concentrations in the mother [14,15]. The glycine–glutamate turnover cycle between the fetal circulation and placenta has been well described. Glycine is transported from the mother into the fetal circulation by active transport, and is converted to glutamate predominantly in the fetal liver. When excess glutamate is present in the fetal circulation, the glutamate is transported to the placenta to prevent glutamateinduced neurotoxicity. The placenta is responsible for the metabolism of as much as 80% of fetal glutamate [13,14]. Based on our prior studies with blood glutamate scavengers GOT, GPT, oxaloacetate, and pyruvate, we theorize that fetal blood glutamate concentrations may potentially be reduced via a reduction of maternal blood glutamate levels. If this is indeed possible, this decrease in fetal blood glutamate concentrations would be expected to result in the fast transport of excess glutamate from the fetal brain to the blood, thereby providing neuroprotection. This is especially important because this would provide the potential to begin early treatment in utero, with the continuation of treatment after delivery. The purpose of this study was to investigate the relationship between concentrations of glutamate in the maternal blood and fetal blood (umbilical artery and vein), to determine the concentrations of GOT and GPT and their correlation with glutamate levels, and to determine the relationship between fetal glutamate levels and fetal outcomes. 2. Materials and methods This experiment has been conducted according to the recommendations set by the Helsinki Committee and was approved by the Ethics Committee of Ben Gurion University of the Negev, Beer Sheva, Israel. Each participant signed an informed consent before the beginning of the experiment. 2.1. Studied population A total of 87 pregnant females between the ages of 18 and 40 participated in the study. For the purpose of determining a correlation between blood glutamate, GOT and GPT concentrations in different conditions, mothers were included in the study irrespective to any coexisting pathology, method of delivery (cesarean section or vaginal), or type of anesthesia or analgesia in cases of cesarean section or vaginal delivery, respectively. 2.2. Experimental design Enrolled mothers in the study were followed up during their labor. Immediately after delivery and the clamping of the umbilical cord, blood samples were extracted simultaneously from the maternal peripheral vein, umbilical artery and umbilical vein into pre-marked syringes. Volumes of each blood sample were measured at approximately 5 ml. Immediately after blood samples collection, 0.2 ml of blood was removed from each blood sample for the determination of glutamate levels in whole blood, as described below. The remainder of each blood sample was used to measure serum GOT and GPT concentrations, as described below. The following data was collected from medical records: age of parturients, age of the pregnancy, symptoms of fetal distress (listed below), sex of newborns, co-existing conditions (both maternal and fetal) and method of delivery (vaginal or cesarean section).
Fetal distress was considered when at least one of the following symptoms was registered during labor and immediately after delivery: fetal bradycardia (defined as fetal heart rate by monitor b 100 BPM), fetal tachycardia (defined as fetal heart rate by monitor > 160 BPM), late decelerations, variable decelerations, meconial, infectious or bloody amniotic fluids, Apgar score ≤ 7 in the first minute, necessity of newborn intubation for oxygenation maintenance, or umbilical artery blood pH on delivery ≤ 7.2. 2.3. GOT and GOT determination in serum Serum GOT and GPT levels were determined in the biochemical laboratory of Soroka Medical Center via a fluorescent method, based on the conversion of glutamate into alanine and aspartate in the presence of GOT and GPT respectively (Olympus AU 2700). 2.4. Glutamate determination in whole blood Whole blood (200 μl aliquot) was deproteinized by adding an equal volume of ice-cold 1 M perchloric acid (PCA) and then centrifuging at 10,000 ×g for 10 min at 4 °C. The pellet was discarded and 200 μl supernatant collected, adjusted to pH 7.2 with 50 μl 2 M K2CO3, repeatedly centrifuged at 10,000 ×g for 4 min at 4 °C and stored at −80 °C for later analysis (at a REVCO freezer). Glutamate concentrations were measured using the fluorometric method of Graham and Aprison [16]. A 20 μl aliquot from the PCA supernatant was added to 480 μl of a 0.3 M glycine, 0.25 M hydrazine hydrate buffer adjusted to pH 8.6 with 1 N H2SO4 and containing 15 U of glutamate dehydrogenase in 0.2 mM NAD. After incubation for 30–45 min at room temperature, the fluorescence was measured at 460 nm with excitation at 350 nm. A glutamate standard curve was established with concentrations ranging from 0 to 6 μM. All determinations were done at least in duplicates. 2.5. Statistics Levels of glutamate, GOT and GPT were compared between maternal, umbilical artery and umbilical vein samples using repeated measures analysis of variance with Bonferroni post‐hoc testing. Pearson's correlation coefficient was used to analyze the relationship between concentrations of glutamate, GOT and GPT in maternal, umbilical artery and umbilical vein blood. Statistical analysis was performed by using the SPSS 18.0 software package (SPSS, Inc, Chicago, IL). Differences in data were considered significant when p b 0.05 and highly significant when p b 0.01. Data are presented as average ± SEM. 3. Results 3.1. Subjects A total of 87 pregnant females between the ages of 18 and 40 participated in the study. The average maternal age was 26 ± 9 years. The average age of pregnancy was 38 ± 3 weeks. Of the 87 delivered newborns, 41 were females and remaining 46 were males. Of the 87 participants, we were unable to collect a blood sample from the umbilical artery in 8 women and were unable to collect a blood sample from the umbilical vein in 3 women. This was due to small volumes of blood in the artery or vein respectively or due to early clot formation. Fifty-five participants underwent a vaginal delivery. The remaining 32 delivered via cesarean section, of which 23 were anesthetized via general anesthesia and 9 were anesthetized with spiral or epidural anesthesia. General anesthesia included induction via preoxygenation, 2–3 mg/kg of propofol, and 1.5 mg/kg succinylcholine, with maintenance via isoflurane 1 MAC, N2O/O2 50:50, 1–2 μg/kg of fentanyl and either atracurium or rocuronium was used for muscle relaxation.
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3.2. Blood glutamate concentrations Fetal blood glutamate concentrations, in both the umbilical artery and vein, were nearly twice as high as maternal blood glutamate concentrations (p b 0.001, Fig. 1). There were no significant differences between glutamate concentrations in the umbilical artery and umbilical vein. There was an association observed between glutamate levels in maternal blood and glutamate levels in both venous (R = 0.32, p b 0.01) and arterial fetal blood (R = 0.33, p b 0.05). There was a significant positive Pearson's correlation found between blood glutamate concentrations in arterial and venous fetal blood (R = 0.85, p b 0.001) (Fig. 2). 3.3. Serum GOT and GPT levels Measured serum GOT and GPT levels are presented in Table 1. Maternal serum GOT levels were found to be significantly lower than fetal serum GOT levels in the umbilical artery and vein (pb 0.001). There were no significant difference between GOT concentrations in umbilical artery and umbilical vein. On the contrary, GPT levels did not differ significantly between maternal serum, umbilical artery and umbilical vein. There was no significant correlation found between GOT or GPT levels in maternal and fetal serum. However, there was a strong correlation found between levels of GOT in arterial and venous fetal serum (R = 0.72, p b 0.001). 3.4. Correlation between symptoms of fetal distress and fetal blood glutamate levels There were 21 cases where the fetus or newborn presented with at least one of the symptoms of fetal distress listed above. Of them, meconial amniotic fluid was seen in 6 cases, a low Apgar score was observed in 5 cases, and a low pH was measured in 4 cases. Severe distress, based on an Apgar score of ≤7 in the first minute with concurrent umbilical artery blood pH on delivery of ≤7.2, was seen in only 4 cases. Five babies were diagnosed with intrauterine growth retardation at birth. There was no difference in glutamate levels found between those with or without fetal distress in either the umbilical artery or umbilical vein.
Fig. 1. Differences in blood glutamate concentrations. Fetal blood glutamate concentrations were found to be significantly higher than maternal blood levels in both the umbilical artery and vein (p b 0.001 *). There were no significant differences between the glutamate concentrations in the fetal umbilical artery and umbilical vein. Data are presented as means ± SEM.
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3.5. Correlation between gender of newborns and blood glutamate, GOT and GPT levels Forty-one of the 87 delivered newborns were female, and the remaining 46 were male. There were no statistically significant differences in blood glutamate levels between female and male newborns in arterial umbilical blood or venous umbilical blood. There were no significant differences in serum GOT levels between female and male newborns. There were no significant differences in serum GPT levels between female and male newborns. 4. Discussion In this study we demonstrated that glutamate concentrations in whole blood were significantly higher in both the umbilical arterial and venous blood compared with maternal blood. GOT levels were significantly higher in the umbilical arterial and venous serum compared with maternal serum, while GPT levels were not significantly different. The concentration of glutamate in fetal blood correlated with the glutamate concentration in maternal blood. There was no difference in fetal glutamate, GOT or GPT between the umbilical artery and vein. It was previously shown that the concentrations of many amino acids in fetal plasma exceed the concentrations in maternal plasma [15]. The high glutamate concentrations in fetal blood relative to maternal blood, as was demonstrated in this study, may not be due to a limited ability to clear glutamate. Rather, these higher concentrations likely reflect an important role for normal brain development. Excitatory amino acids were shown to be crucial in the development of brain plasticity in animals and humans, and the expression of glutamate receptors is enhanced in the developing animal and human brain. It has further been shown that plasma glutamate concentrations are maximally elevated in newborns, rapidly decrease during the first months of life, and then slowly decreases over the following several years [17,18]. As such, NMDA receptor blockade significantly influences the development of cortical plasticity [19]. Although significantly elevated glutamate levels after brain insults are implicated in secondary brain injury, NMDA receptor antagonists failed to provide neuroprotection in human studies with both TBI and stroke [20]. One possible explanation for this may be that glutamate, at normal concentrations in the brain's fluids, is critical for preserving normal neuronal function by activating glutamate receptor signaling (via both ionotropic and metabotropic receptors) and maintaining neuronal integrity. NMDA receptor antagonists interfere with both the negative and positive effects of this signaling [21,22]. Therefore, glutamate levels in both the brain's ECF and the blood require tight regulation to balance its positive and neurotoxic effects. It is known that GOT, an enzyme present in the blood, converts glutamate to its non-toxic metabolite 2-ketoglutarate. In this study, we observed that GOT levels in fetal serum were higher than maternal GOT levels. As expected from the Michaelis–Menten enzyme rate equation, higher levels of GOT are necessary to convert the increased amount of glutamate in fetal blood into 2-ketoglutarate. Our finding that the elevated concentration of GOT in fetal plasma directly correlates with the rise in glutamate suggests that the elimination of glutamate does not rely solely on the placenta. Rather, this elevation of GOT is likely necessary to maintain the adequate balance between the positive and negative effects of glutamate on the developing brain [19]. Hays and colleagues showed that dietary glutamate is almost entirely removed from the bloodstream via first pass metabolism through the splanchnic bed in premature infants [23]. This is not surprising considering the very high concentrations of GOT in newborns' serum, supporting the idea that the elevated GOT levels have high glutamate-converting capacity in the context of elevated blood glutamate levels. Conversely, GPT levels were not elevated in fetal serum, suggesting that the GPT-dependent mechanism of glutamate
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Fig. 2. The correlations between blood glutamate and GOT concentrations. The correlation between glutamate levels in maternal blood and glutamate levels in arterial fetal blood was R = 0.33 (p b 0.05; panel A). The correlation between glutamate levels in maternal blood and glutamate levels in venous fetal blood was R = 0.32 (p b 0.01; panel B).The correlation between blood glutamate concentrations in arterial and venous fetal blood was R = 0.85 (p b 0.001; panel C). The correlation between GOT in arterial and venous fetal serum was R = 0.72 (p b 0.001; panel D).
metabolism plays a less significant role than GOT. The limited significance of GPT compared with GPT reflects previous observations in adult humans after ischemic stroke [10–12] and in rats after TBI [24]. The literature regarding glutamate concentrations in fetal or newborns’ plasma compared to maternal concentrations is inconsistent. Cetin and colleagues reported that glutamate concentrations in the umbilical artery is 1.5 times lower than in the maternal plasma, while glutamate concentrations in the umbilical vein were two-fold lower than in the umbilical artery [14]. However, the authors employed Table 1 Measured serum GOT and GPT concentrations. Fetal serum GOT concentrations were found to be significantly higher than maternal serum levels in both the umbilical artery and vein (p b 0.001 *). The difference in GPT levels between maternal and fetal blood was not statistically significant. There was no difference in fetal GOT or GPT between the umbilical artery and vein. Data are presented as means ± SEM. Parameter
Maternal blood
Umbilical artery
Umbilical vein
Number of samples analyzed GOT (U/L) GPT (U/L)
87 19 ± 1 11 ± 1
79 39* ± 2 13 ± 1
84 38* ± 2 13 ± 1
a much smaller sample size than that of our study, and the discrepancy may be due to differences in the study design. Furthermore, while their study measured glutamate levels in maternal and fetal plasma, our findings reflect that of whole blood. Considering that the concentration of glutamate in erythrocytes is 4 to 5 times higher than in plasma [25], and that hemoglobin and hematocrit concentrations in the fetus and newborn are almost two times higher than in pregnant women, fetal whole blood has a much higher concentration of glutamate compared with maternal whole blood. Moreover, two other studies demonstrated that the concentration of glutamate in the plasma during one's lifetime is maximal in newborns, rapidly decreases during the first months of life, and then slowly decreases over the following several years [17,18]. If this is the case, it would not be possible for fetal or newborn blood glutamate levels to be lower than maternal levels. We demonstrated an association between maternal and fetal blood glutamate levels. This is an especially important finding because it suggests that under fetal asphyxia conditions, one may theoretically reduce fetal blood glutamate concentrations by decreasing maternal blood glutamate levels. However, causality in the observed association between
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maternal and fetal glutamate, GOT and GPT levels cannot yet be presumed and additional experimental studies are still necessary to test this hypothesis. Yet, these findings may initiate future studies to explore the possibility of new treatment strategies directed at decreasing glutamate concentration in the fetal brain, promoting neuroprotection without affecting the vitally important functions of glutamate in the developing brain. There were no differences observed in fetal glutamate, GOT or GPT between the umbilical artery and vein. Glutamate is an amino acid that is intensively metabolized and synthesized in all compartments of the body: blood, peripheral tissues and placenta. A free shift of unchanged glutamate exists between these different compartments. In adults, for example, under normal conditions, blood glutamate levels are in a steady state regulated by a net export from the liver and a net utilization by the skeletal muscles and gut [26]. Therefore, considering this very complex mechanism of glutamate's metabolism with the concurrent synthesis and distribution in different tissues, one would assume different glutamate concentrations between venous and arterial blood in the fetus. Therefore, this observation was not as expected and is an important finding. We found no differences in blood glutamate levels between newborns with symptoms of fetal distress and those without symptoms of fetal distress. This finding was not surprising, for if fetal distress and asphyxia affects fetal blood glutamate levels, it is reasonable that this process would take at least several hours to develop. Today, it is common in obstetric practice to monitor the fetal condition during labor and respond immediately if symptoms of fetal distress appear. Usually, within a few minutes of the development of symptoms of fetal distress, the fetus is delivered via cesarean section or vaginally with a vacuum. However, in this study, there were a limited number of cases in which fetal distress was present precluding us from making any final conclusions regarding this issue. We previously showed that the female sex hormones estrogen and progesterone were inversely correlated with blood glutamate levels [27,28]. As such, in human studies blood glutamate and GOT levels were shown to be much lower in females than in males [27,28]. For this reason, we examined whether gender differences in blood glutamate levels exist in newborns. We did not find any significant differences in blood glutamate, GOT and GPT levels between female and male newborns. This may be explained by an equal concentration of female sex hormones in the male and female fetus and newborn due to an immaturity of their gonadal glands [29]. Considering that the placenta has a well-developed system of transporters for glutamate elimination, it seems plausible that one may create a favorable chain of glutamate concentration gradients that would ultimately lead to the elimination of excess glutamate from the fetal brain. This chain would start with the reduction of glutamate concentrations in the maternal blood compartment, thereby creating a favorable glutamate gradient between the maternal and fetal blood. This would lead to a placenta-mediated efflux of glutamate from the fetal to maternal blood. Decreases in fetal blood glutamate concentrations should then facilitate the efflux of excess of glutamate from the fetal brain's ECF compartment, thereby preventing its neurotoxic effects. The results of this study may offer new therapeutic insights into future treatment modalities of fetal blood glutamate reduction in utero by way of reducing maternal glutamate levels. In newborns, asphyxia has been shown to be associated with an elevated ECF glutamate concentration that slowly decreases toward baseline levels by 72 h [1]. Considering that fetal distress may be diagnosed early during labor via fetal monitoring, prompt initiation of the treatment may improve neurological outcome in newborns. While the efficacy of glutamate-reducing therapeutic options is not yet clear, potentially such treatment could be initiated “in utero” and continued after delivery. Additional experiments in both clinical and experimental settings are still warranted to investigate potential treatment strategies and their therapeutic window.
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In conclusion, we demonstrated that higher baseline concentrations of blood glutamate are present in fetal blood compared with maternal blood, and this was associated with an increase in GOT, but not GPT. An association was observed between maternal and fetal blood glutamate levels. These findings are important in investigating the possibility of new therapeutic strategies for fetal asphyxia conditions, via decreasing fetal blood glutamate concentrations in utero by reducing maternal glutamate levels.
Conflict of interest statement The authors state that no competing financial or other conflicts of interests exist.
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