细菌性脑膜炎患儿脑脊液中兴奋性氨基酸的变化及意义
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摘要
目的 观察细菌性脑膜炎(BM)患儿脑脊液(CSF)中兴奋性氨基酸(EAA)谷氨酸(Glu)和天门冬氨酸(Asp)含量的变化，并探讨其在细菌性脑膜炎病理生理过程中的作用和临床意义。
方法 临床诊断为细菌性脑膜炎的40例患儿根据中枢神经系统功能障碍的程度分为轻度组(12例)和重度组(28例)，16例无神经系统疾病但具有腰穿指征的同期住院患儿为对照组，其中急性白血病(缓解期)8例，鼻窦炎5例，维生素D缺乏性手足搐搦症3例。所有患儿于入院当天应用抗菌素治疗前做腰穿留取脑脊液进行常规、生化及细菌学检查，另取脑脊液1-2ml立即置-30℃冰箱中保存用于Glu和Asp的测定。细菌性脑膜炎患儿于抗菌治疗三周后再次留取脑脊液复查常规、生化及细菌学检查，另取脑脊液标本冷冻待测。有神经系统后遗症的患儿出院三个月后复诊时第三次留取脑脊液标本，所有脑脊液标本详细标号登记后立即置-30℃冰箱中冷冻待测。实验使用日本岛津LC-6A高效液相色谱仪，采用OPA(邻苯二甲醛)、2-ME(2-巯基乙醇)柱前衍生氨基酸HPLC检测法测定CSF中Glu、Asp的浓度。
结果1.细菌性脑膜炎组CSF中Glu、AsP浓度明显高于对照组
(P值分别小于0.001和0.05)2.秉度细菌性脑膜炎患儿脑脊液中
以u、AsP浓度明显高于轻度组患儿(P均小于0.001) 3.32例细菌
性脑膜炎患儿经三周抗菌治步犷后8例病人CSF中Glu、AsP浓度持
续升高，其中7例留有智力障碍、脑瘫、癫痛等神经系统后遗症，
而另24例病人脑脊液中Glu、AsP含量较入院时明显下降，其中仅
2人留有后遗症，经卡方检验者有显著差异性(犷=6.095，P<0.05)，
6例有后遗症的患儿二个月后复诊时仍被发现脑脊液中Glu、AsP
含量高于对照组水平。4.细菌性脑膜炎患儿CSF中Glu、AsP浓度
‘J CSF中蛋白质含量呈正相关(r值分别为0.885和0.773，P值均
小于0.001)，而与葡萄糖含量呈负相关(r值分别为一0.660和一0.615，
P值均小于0.0])。
结论1.兴奋性氨基酸参与了细菌性脑膜炎的病理生理过程。
2.测定CSF中兴奋性氨基酸浓度对细菌性脑膜炎病情严一甭程度的
判断具有指一导意义。3.脑脊液中兴奋性氨基酸持续升高可以预，J之细
菌性脑膜炎病人预后较差，日_兴奋毒作用较长时问地存在于有补}，经
系统后遗症的病人。4.脑脊液中兴奋性氨基酸浓度的测定是评价细
菌性脑膜炎脑损伤程度敏感和可靠的生化指标，i犷为EAA受体拮
抗剂的临床应用提供了可靠的临床实验依据。
Objectives To study the change of excitatory amino acids (EAA) glutamate (Glu) and aspartic (Asp) in cerebrospinal fluid (CSF)of children with bacterial meningitis and their function in pathophysiological mechanisms and clinical significance.
Methods Aaccording to the clinical diagnosis criterion 40 patients of bacterial meningitis were chosen and classified into two groups, 28 cases of mild state, 12 cases of severe state , the clinical severity of the disease was assessed on admission. CSF control samples were obtained from 16 children without neurogenic diseases. The CSF samples were obtained by lumber puncture on admission before the beginning of antibiotic treatment and on the 21st day after the beginning of antibiotic treatment. The third group CSF samples were gotten from those who had poor clinical outcome three months since they were discharged from hospital. In addition to standard CSF investigations, including cell count ,cyctologic findings, protein analysis , glucose levels, the
concentration of glutamate and aspartate in the CSF was measured. All CSF samples were marked conscientiously and frozen immediately and stored for use at -30 in a refrigerator. The concentration of excitatory amino glutamate and aspartate was measured by high-performance liquid chromatography.
Results 1. The CSF levels of excitatory amino glutamate and aspartate of children with bacterial meningitis were significantly higher than those of the controls (P<0.001 and 0.05). 2.The CSF levels of excitatory amino acid of patients with severe state were increased than those of mild state (P<0.001) 3. After 21 days antibiotic treatment , the concentration of excitatory amino acids were decreased significantly in 24 patients. However, in 8 patients the levels remained higher and their clinical outcomes were significantly worse than those of lower levels in the second CSF analysis(x2=6.095, P<0.05). The concentration of glutamate and aspartate of those who had poor clinical outcome three months since they were discharged from hospital did not decreased the normal levels. 4.The CSF levels of excitatory amino acid were negatively correlated with the concentrations of CSF glucose and positively correlated with the concentrations of protein in CSF.
Conclusions 1. The changing of EAA in CSF in children with bacterial meningitis indicates that they may participate in pathophysiological mechanisms of BM. 2.Testing the levels of EAA will contribute to the judgment of the severity of cerebral injury of BM.
3. A prolonged increase of EAA levels in the CSF may predict poor clinical outcome in patients with BM.
Our findings showed that the concentrations of EAA in CSF are sensitive and reliable biochemical markers for evaluating the extent of brain damage of patients with bacterial meningitis and testing EAA concentration provided helpful clinical study data to us for the clinical use of the inhibitor of EAA receptor.
方法 临床诊断为细菌性脑膜炎的40例患儿根据中枢神经系统功能障碍的程度分为轻度组(12例)和重度组(28例)，16例无神经系统疾病但具有腰穿指征的同期住院患儿为对照组，其中急性白血病(缓解期)8例，鼻窦炎5例，维生素D缺乏性手足搐搦症3例。所有患儿于入院当天应用抗菌素治疗前做腰穿留取脑脊液进行常规、生化及细菌学检查，另取脑脊液1-2ml立即置-30℃冰箱中保存用于Glu和Asp的测定。细菌性脑膜炎患儿于抗菌治疗三周后再次留取脑脊液复查常规、生化及细菌学检查，另取脑脊液标本冷冻待测。有神经系统后遗症的患儿出院三个月后复诊时第三次留取脑脊液标本，所有脑脊液标本详细标号登记后立即置-30℃冰箱中冷冻待测。实验使用日本岛津LC-6A高效液相色谱仪，采用OPA(邻苯二甲醛)、2-ME(2-巯基乙醇)柱前衍生氨基酸HPLC检测法测定CSF中Glu、Asp的浓度。
结果1.细菌性脑膜炎组CSF中Glu、AsP浓度明显高于对照组
(P值分别小于0.001和0.05)2.秉度细菌性脑膜炎患儿脑脊液中
以u、AsP浓度明显高于轻度组患儿(P均小于0.001) 3.32例细菌
性脑膜炎患儿经三周抗菌治步犷后8例病人CSF中Glu、AsP浓度持
续升高，其中7例留有智力障碍、脑瘫、癫痛等神经系统后遗症，
而另24例病人脑脊液中Glu、AsP含量较入院时明显下降，其中仅
2人留有后遗症，经卡方检验者有显著差异性(犷=6.095，P<0.05)，
6例有后遗症的患儿二个月后复诊时仍被发现脑脊液中Glu、AsP
含量高于对照组水平。4.细菌性脑膜炎患儿CSF中Glu、AsP浓度
‘J CSF中蛋白质含量呈正相关(r值分别为0.885和0.773，P值均
小于0.001)，而与葡萄糖含量呈负相关(r值分别为一0.660和一0.615，
P值均小于0.0])。
结论1.兴奋性氨基酸参与了细菌性脑膜炎的病理生理过程。
2.测定CSF中兴奋性氨基酸浓度对细菌性脑膜炎病情严一甭程度的
判断具有指一导意义。3.脑脊液中兴奋性氨基酸持续升高可以预，J之细
菌性脑膜炎病人预后较差，日_兴奋毒作用较长时问地存在于有补}，经
系统后遗症的病人。4.脑脊液中兴奋性氨基酸浓度的测定是评价细
菌性脑膜炎脑损伤程度敏感和可靠的生化指标，i犷为EAA受体拮
抗剂的临床应用提供了可靠的临床实验依据。
Objectives To study the change of excitatory amino acids (EAA) glutamate (Glu) and aspartic (Asp) in cerebrospinal fluid (CSF)of children with bacterial meningitis and their function in pathophysiological mechanisms and clinical significance.
Methods Aaccording to the clinical diagnosis criterion 40 patients of bacterial meningitis were chosen and classified into two groups, 28 cases of mild state, 12 cases of severe state , the clinical severity of the disease was assessed on admission. CSF control samples were obtained from 16 children without neurogenic diseases. The CSF samples were obtained by lumber puncture on admission before the beginning of antibiotic treatment and on the 21st day after the beginning of antibiotic treatment. The third group CSF samples were gotten from those who had poor clinical outcome three months since they were discharged from hospital. In addition to standard CSF investigations, including cell count ,cyctologic findings, protein analysis , glucose levels, the
concentration of glutamate and aspartate in the CSF was measured. All CSF samples were marked conscientiously and frozen immediately and stored for use at -30 in a refrigerator. The concentration of excitatory amino glutamate and aspartate was measured by high-performance liquid chromatography.
Results 1. The CSF levels of excitatory amino glutamate and aspartate of children with bacterial meningitis were significantly higher than those of the controls (P<0.001 and 0.05). 2.The CSF levels of excitatory amino acid of patients with severe state were increased than those of mild state (P<0.001) 3. After 21 days antibiotic treatment , the concentration of excitatory amino acids were decreased significantly in 24 patients. However, in 8 patients the levels remained higher and their clinical outcomes were significantly worse than those of lower levels in the second CSF analysis(x2=6.095, P<0.05). The concentration of glutamate and aspartate of those who had poor clinical outcome three months since they were discharged from hospital did not decreased the normal levels. 4.The CSF levels of excitatory amino acid were negatively correlated with the concentrations of CSF glucose and positively correlated with the concentrations of protein in CSF.
Conclusions 1. The changing of EAA in CSF in children with bacterial meningitis indicates that they may participate in pathophysiological mechanisms of BM. 2.Testing the levels of EAA will contribute to the judgment of the severity of cerebral injury of BM.
3. A prolonged increase of EAA levels in the CSF may predict poor clinical outcome in patients with BM.
Our findings showed that the concentrations of EAA in CSF are sensitive and reliable biochemical markers for evaluating the extent of brain damage of patients with bacterial meningitis and testing EAA concentration provided helpful clinical study data to us for the clinical use of the inhibitor of EAA receptor.
引文
1 许绍芬．神经生物学．第2版．上海：上海医科大学出版社，1999，208-209．
2 Olney, JW, HO OC, Rheev. Cvtotoxic effects of acidic Sulphercontaining amino acids on the infant mouse central nervous system. Exp BrainRes, 1971, 14(1):61.
3 Choi DW, Rothman SM. The role of glutomate neurotoxicity in hypoxic-ischemic neuronal death. Ann Rev Neurosci, 1990, 18:171-182.
4 Greenamyne JT, Porter Hp. Anatomy and physiology of glutmate in the CNS. Neurology, 1994, 44 Suppl 8:s7-s13.
5 Chakvo M, Kalincakova K,, Kluchova D, et al. Blood flow and electrytes in spinal cord ischemia. Exp Neurol, 1991, 112(3):299.
6 Greenamyer JY, Greene JT, Higgins DS, et al. Mechanism of selective neuronal vulnerability: Bioenergetics and excitotox icity, In: Marwark J, Prasad KN, Teitelbaum H, eds. Neural Transpl antation, CNS Neuronal Injury, and Regen-
eration. New York:CRC Press, 1994, 71-80.
7 Choi DW, Excitotoxic cell death. J Neurobiol, 1992, 23: 1261-1276.
8 Olney JW, Wozniak DF, Farber NB. Excitotoxic neurodegeneration in Alzheimer disease. New hypothesis and new therapeutic strategies. Arch Neurol, 1997, 54:1234-1240.
9 Guerra-Romero-L, Tureen-J-H, Fournier-M-A. Amino acids in cerebrospinal and brain interstitial fluid in experimental pneumococcal meningitis. Pediatr-Res, 1993, 33(5):510-513.
10 李菊，杨期东．微透析技术和丹参注射液对Wistar鼠脑缺血时海马细胞外液兴奋性氨基酸释放的影响．中风与神经疾病杂志．1995，12：258-260．
11 Baker AJ, Moulton RJ, Macmillan VH, et al. Excitatory amino acids in cerebrospinal fluid following traumatic brain injury in human. J Neurosury, 1993, 79:369-372.
12 Begley DJ, Reichel A, Ermisch A. Simple high-performance liquid chromatographic analysis of free primary amino acid concentrations in rat plasma and cisternal cerebrospinal fluid. J Chromatogr B Biomed Appl, 1994, 657:185-191.
13 丁敏，曾成鸣．柱前衍生高效液相色谱法分析脑脊液氨基酸．上海医学检验杂志，1995，20：24-25．
14 张虹，翟所迪，李家仁．脑外伤脑脊液中兴奋性氨基酸测定及意义．中华医学检验杂志，1998．6：359-361．
15 尹飞，杨于嘉，虞佩兰等．百日咳菌液诱发大鼠水肿时脑组织中兴奋性氨基酸时相的变化．中华儿科杂志，2000，38(1)：19-12．
16 Leib-SL, Kim-YS, Ferriero-DM, etal. Neuroprotective effect of excitatory amimo acid antagonist kynurenic in experimental bacterial meningitis. J-Infect-Dis, 1996, 173 (1):166-171.
17 王慕逖．儿科学(第5版)．北京：人民卫生出版社．1998，392-395．
18 金汉珍，黄德珉，官希吉．实用新生儿学(第2版)．北京：人民卫生出版社．1990，293-294．
19 Guerra-Romero L,Tureen JH, Fournier MA, et al. Amino acids in cerebrospinal and interstitial fluid in experimental pneumococcal meningitis. Pediatr Res, 1993, 33:510-513.
20 Spranger-M, Krempion-S, Schwab-S, et al. Excess glutamate in the cerebrospinal fluid in bacterial meningitis. J-Neuro-Sci.
1996, Nov, 143:126-131.
21 Kornelisse RF. The role of nitric oxide in bacterial meningitis in children. J Infect Dis. 1996, 174: 120-126.
22 许丽艳．一氧化氮对体外培养大鼠脑神经细胞的神经毒作用的初探．中风与神经疾病杂志，1994，11(6)：339-341．
23 吴希如，林庆．小儿神经系统疾病基础与临床．北京：人民卫生出版社．2000，386-389．
24 Nau, -Roland; Bruck, -Wolfgang. Neuronal in jury in bacterial meningitis: mechanisms and implications for therpy. Trends-Neurosci, 2002, 25(1):38-45.
25 韩玉锟，许植之，虞人杰．新生儿缺氧缺血性脑病．北京：人民卫生出版社．2000，49-50．
26 Wenthold RJ, Altschulter RA, Skaggs KK, et al. Immunochemical characterization of glutamate dehydrogenase in the cerebellum of the rat. J. Neurochem, 1987, 48:636.
27 Perry-V-L, Young-R-S, Aquila-W-J. Effect of experimental Escherichia coli meningitis on concentrations of excitatory and inhibitory amino acids in the rabbit brain in vivo microdialysis
study. Pediatr-Res, 1993, 34(2):187-191.
28 Spranger-M, Schwab-S, Krempion-S, et al. Excess glutamate levels in the cerebrospinal fluid predict clinical outcome of bacterial meningitis. Arch-Neurol, 1996, Oct, 56 (10): 992-996.
29 Gwag BJ, Koh JY, Demaro Ji, et al. Slowly triggered excitoxicity occurs by necrosis in cortical cultures. Neuroscience, 1997, 77(2): 393-401.
30 heng Y, Deshmukh M, D' Costa A, et al. Caspase inhibitor Affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J clin Invest, 1998, 101 (9): 1992-1999.
31 Du Y, Dodel RC, Bales KR, et al. Involvement of caspase-3-like cystetine protease in 1-methyI-4phenyIpyridinium-mediated apotosis of cultured cerebellar granule neurons. J Neurochem, 1997, 69(4): 1382-1388.
32 Dugan LL, Sensi SL, Canzonier LMT, etal. Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methy-D-aspartate. J Neurosci,
1995，15(10)：6377-6388．
33 肖镇祥．临床脑脊液学．北京：人民卫生出版社．1982，82-102．
2 Olney, JW, HO OC, Rheev. Cvtotoxic effects of acidic Sulphercontaining amino acids on the infant mouse central nervous system. Exp BrainRes, 1971, 14(1):61.
3 Choi DW, Rothman SM. The role of glutomate neurotoxicity in hypoxic-ischemic neuronal death. Ann Rev Neurosci, 1990, 18:171-182.
4 Greenamyne JT, Porter Hp. Anatomy and physiology of glutmate in the CNS. Neurology, 1994, 44 Suppl 8:s7-s13.
5 Chakvo M, Kalincakova K,, Kluchova D, et al. Blood flow and electrytes in spinal cord ischemia. Exp Neurol, 1991, 112(3):299.
6 Greenamyer JY, Greene JT, Higgins DS, et al. Mechanism of selective neuronal vulnerability: Bioenergetics and excitotox icity, In: Marwark J, Prasad KN, Teitelbaum H, eds. Neural Transpl antation, CNS Neuronal Injury, and Regen-
eration. New York:CRC Press, 1994, 71-80.
7 Choi DW, Excitotoxic cell death. J Neurobiol, 1992, 23: 1261-1276.
8 Olney JW, Wozniak DF, Farber NB. Excitotoxic neurodegeneration in Alzheimer disease. New hypothesis and new therapeutic strategies. Arch Neurol, 1997, 54:1234-1240.
9 Guerra-Romero-L, Tureen-J-H, Fournier-M-A. Amino acids in cerebrospinal and brain interstitial fluid in experimental pneumococcal meningitis. Pediatr-Res, 1993, 33(5):510-513.
10 李菊，杨期东．微透析技术和丹参注射液对Wistar鼠脑缺血时海马细胞外液兴奋性氨基酸释放的影响．中风与神经疾病杂志．1995，12：258-260．
11 Baker AJ, Moulton RJ, Macmillan VH, et al. Excitatory amino acids in cerebrospinal fluid following traumatic brain injury in human. J Neurosury, 1993, 79:369-372.
12 Begley DJ, Reichel A, Ermisch A. Simple high-performance liquid chromatographic analysis of free primary amino acid concentrations in rat plasma and cisternal cerebrospinal fluid. J Chromatogr B Biomed Appl, 1994, 657:185-191.
13 丁敏，曾成鸣．柱前衍生高效液相色谱法分析脑脊液氨基酸．上海医学检验杂志，1995，20：24-25．
14 张虹，翟所迪，李家仁．脑外伤脑脊液中兴奋性氨基酸测定及意义．中华医学检验杂志，1998．6：359-361．
15 尹飞，杨于嘉，虞佩兰等．百日咳菌液诱发大鼠水肿时脑组织中兴奋性氨基酸时相的变化．中华儿科杂志，2000，38(1)：19-12．
16 Leib-SL, Kim-YS, Ferriero-DM, etal. Neuroprotective effect of excitatory amimo acid antagonist kynurenic in experimental bacterial meningitis. J-Infect-Dis, 1996, 173 (1):166-171.
17 王慕逖．儿科学(第5版)．北京：人民卫生出版社．1998，392-395．
18 金汉珍，黄德珉，官希吉．实用新生儿学(第2版)．北京：人民卫生出版社．1990，293-294．
19 Guerra-Romero L,Tureen JH, Fournier MA, et al. Amino acids in cerebrospinal and interstitial fluid in experimental pneumococcal meningitis. Pediatr Res, 1993, 33:510-513.
20 Spranger-M, Krempion-S, Schwab-S, et al. Excess glutamate in the cerebrospinal fluid in bacterial meningitis. J-Neuro-Sci.
1996, Nov, 143:126-131.
21 Kornelisse RF. The role of nitric oxide in bacterial meningitis in children. J Infect Dis. 1996, 174: 120-126.
22 许丽艳．一氧化氮对体外培养大鼠脑神经细胞的神经毒作用的初探．中风与神经疾病杂志，1994，11(6)：339-341．
23 吴希如，林庆．小儿神经系统疾病基础与临床．北京：人民卫生出版社．2000，386-389．
24 Nau, -Roland; Bruck, -Wolfgang. Neuronal in jury in bacterial meningitis: mechanisms and implications for therpy. Trends-Neurosci, 2002, 25(1):38-45.
25 韩玉锟，许植之，虞人杰．新生儿缺氧缺血性脑病．北京：人民卫生出版社．2000，49-50．
26 Wenthold RJ, Altschulter RA, Skaggs KK, et al. Immunochemical characterization of glutamate dehydrogenase in the cerebellum of the rat. J. Neurochem, 1987, 48:636.
27 Perry-V-L, Young-R-S, Aquila-W-J. Effect of experimental Escherichia coli meningitis on concentrations of excitatory and inhibitory amino acids in the rabbit brain in vivo microdialysis
study. Pediatr-Res, 1993, 34(2):187-191.
28 Spranger-M, Schwab-S, Krempion-S, et al. Excess glutamate levels in the cerebrospinal fluid predict clinical outcome of bacterial meningitis. Arch-Neurol, 1996, Oct, 56 (10): 992-996.
29 Gwag BJ, Koh JY, Demaro Ji, et al. Slowly triggered excitoxicity occurs by necrosis in cortical cultures. Neuroscience, 1997, 77(2): 393-401.
30 heng Y, Deshmukh M, D' Costa A, et al. Caspase inhibitor Affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J clin Invest, 1998, 101 (9): 1992-1999.
31 Du Y, Dodel RC, Bales KR, et al. Involvement of caspase-3-like cystetine protease in 1-methyI-4phenyIpyridinium-mediated apotosis of cultured cerebellar granule neurons. J Neurochem, 1997, 69(4): 1382-1388.
32 Dugan LL, Sensi SL, Canzonier LMT, etal. Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methy-D-aspartate. J Neurosci,
1995，15(10)：6377-6388．
33 肖镇祥．临床脑脊液学．北京：人民卫生出版社．1982，82-102．