Kynurenic acid is decreased in cerebrospinal fluid of patients with infantile spasms

Original Articles

Kynurenic Acid Is Decreased in Cerebrospinal Fluid of Patients With Infantile Spasms Hitoshi Y a m a m o t o , M D , I k u y o Shindo, M D , B u n s e i E g a w a , M D , and Kumiko Horiguchi, MD

Cerebrospinal fluid (CSF) from 8 patients with symptomatic infantile spasms was collected before specific treatment for infantile spasms. The concentration of CSF kynurenic acid (KYA) and 3-hydroxykynurenine (3-OHKY) in infantile spasms was analyzed by highperformance liquid chromatography and compared with CSF KYA from 10 age-matched controls. The levels of CSF KYA were significantly lower in infantile spasm patients compared to controls (P < .05). In contrast, the levels of CSF 3-OHKY were significantly higher in infantile spasm patients than in controls (P < .05). These findings suggest that the presence of seizures in infantile spasms is associated with altered metabolism of 3-OHKY. The possibility that seizures may be related to increased or decreased production of certain kynurenine metabolites is discussed. Y a m a m o t o H, Shindo I, E g a w a B, H o r i g u c h i K. Kynurenic acid is decreased in cerebrospinal fluid of patients with infantile spasms. Pediatr Neurol 1994;10:912.

Introduction Infantile spasms are a form of age-dependent intractable epilepsy in infancy and are often associated with mental retardation [1]. Some evidence suggests that disturbances of brain monoamine activity may play a role in the pathophysiology of the epileptic seizures and sleep disturbances observed during infantile spasms [2,3]. In particular, serotonin neuronal projection abnormalities have been implicated [4]. The kynurenine pathway is a metabolic pathway leading from tryptophan to nicotinamide adenine dinucleotide (Fig 1). Several intermediates of this pathway, including the kynurenines and quinolinic acid (QUIN), have been demonstrated to have various effects on neurotransmission. Recent studies revealed that the kynurenines are neurotoxic [5,6]. In the central nervous system (CNS), kynurenines may modulate excitatory amino acid (EAA)

From the Department of Pediatrics; St. Marianna University School of Medicine; Kawasaki, Japan.

© 1994 by Elsevier Science Inc. • 0887-8994/94/$7.00

transmission and are implicated as neurotoxic agents in the pathogenesis of several neurologic diseases and in seizures [7]. Kynurenic acid (KYA) is the only known endogenous antagonist of EAA receptors and, therefore, may influence important physiologic and pathologic processes [8,9]. QUIN is an intermediate that is present in human brain and that increases in the aging rat brain. QUIN acts as an excitotoxin for specific neuronal groups and produces striatal lesions similar to those observed in some neurologic diseases [10]. EAA-mediated synaptic transmission is the most prevalent excitatory neurotransmitter system in the mammalian brain [ 11]. Activation of EAA receptors has been postulated to contribute to neuronal cell death in epilepsy, hypoglycemia, and Huntington disease [12]; therefore, it would be of interest to determine whether a relationship exists between these endogenous neuromodulators and various neurologic disorders. In our study, the levels of kynurenine metabolites were measured in the cerebrospinal fluid (CSF) of infantile spasm patients and agematched controls before specific treatment for infantile spasms to identify a possible relationship between the symptoms of this disorder and the levels of kynurenine metabolites.

Methods CSF was obtained from 8 infantile spasm patients, ages 5-11 mos (mean: 7.6 mos), and from 10 age-matchedcontrols3-12 mos (mean: 6.1 mos). All patients had markedly abnormal and paroxysmal EEGs. Associated neurologic disorders included perinatal asphyxia, intracranial hemorrhage, tuberous sclerosis, and developmental delay of unknown etiology. All had been treated with antiepileptic drugs for seizures, but none had received treatment specific for infantile spasms such as ACTH, vitamin B6, or prednisone. The clinical data are summarized in Table 1. The diagnosis of infantile spasms was made according to history, physical examination, frequency and type of seizures, and related EEG activity. With the patient in the lateral decubitus position, CSF was collected by lumbar puncture and immediately frozen at -70°C for later analysis. Age-matchedcontrol CSF samples were obtained from patients who required lumbar puncture for diagnosis of fever and were later demonstrated to be normal. None of the controls had any neurologic abnormalities. Analysis of neurotransmitter metabolites was accomplished by direct injection of CSF onto a computer-controlled high-

Communications should be addressed to: Dr. Yamamoto;Department of Pediatrics; St. Marianna University School of Medicine; 2-16-1 Sugao, Miyamae-ku;Kawasaki 216, Japan. Received June 2, 1993; accepted August 30, 1993.

Yamamoto et al: Kynurenic Acid and Spasms 9

tryotamine

B6 -HTP---5-HT--~---~5-HIAA

Figure 1. Summary of metabolic pathways of tryptophan, revealing details of the kynurenine pathway. Underlined compounds were measured in our study. Abbreviations: TRP, tryptophan; 5-HTP, 5-hydroxytryptophan; 5-HT, serotonin; 5-H1AA, 5-hydroxyindoleacetic acid; KYN, kynurenine; 3-OHKY, 3-hydroxykynurenine; XA, xanthurenic acid; 3-OHAN, 3-hydroxyanthranilic acid; QUIN, quinolinic acid: NAMN, nicotinic acid mononucleotide; NAD, nicotinamide adenine dinucleotide ; B 6, vitamin B ~ (pyridoxal phosphate).

Kynurenic acid

KYN ---- 3 -OHKY - - - 3 - O H A N - - - - - - ~ QUIN - - - N A M N B

XA anthranilic acid

performance liquid chromatographic (HPLC) analyzer with electrochemical detector (ESA, Bedford). The concept and inherent advantages of this multielectrode HPLC system have been described previously [13,14]. The gradient profile, column, and detector potential for 3-OHKY detection in this study were similar to those previously described [15]. The mobile phase solutions were obtained from ESA (Bedford) and had the following composition: mobile phase A consisted of 0. l M sodium phosphate with l0 mg/L sodium dodecyl sulfate at pH 3.35; and mobile phase B consisted of 50% methanol/water with 50 mg/L sodium dodecyl sulfate at pH 3.45. Another method, which was isocratic, was developed for the measurement of KYA. The mobile phase consisted of 0.075 M NaH2PO4/0.025 M Na2HPO 4 in 85% methanol. The pH of the mobile phase was adjusted to 6.25 with H2PO4. The electrochemical sensors were set from 0-1,100 mV at 60 mV intervals. KYA was detected predominantly at 1,060 mV. All standards were obtained from Sigma Chemical Company (St. Louis). Significant differences between patients with infantile spasms and controls were examined by the 2-tailed Student t test with unpaired comparison.

Results The concentrations of neurotransmitter metabolites in the CSF of infantile spasm patients before specific treatment for infantile spasms and in CSF samples from agematched controls are presented in Figures 2 and 3. The CSF levels of KYA were significantly lower in infantile spasm patients compared to controls (P < .05). In contrast, the levels of CSF 3-OHKY were significantly higher in infantile spasm patients than in controls (P < .05). These values are presented in Table 2.

Table 1.

Discussion

Our finding of abnormal levels of kynurenine metabolites in the CSF of patients with infantile spasms corroborates other reports of altered monoamine metabolism in this disorder [16]. A major motivation for the study of kynurenines is their possible relevance to seizures. Both 3-OHKY and QUIN are known to produce seizures when injected into rodent brain [17]. KYA is a broad-spectrum EAA receptor antagonist which is present in mammalian CNS [ 18]. Recent evidence suggests that KYA acts at both the glycine allosteric site and the agonist recognition site on the N-methyl-D-aspartate (NMDA) receptor complex. as well as at non-NMDA receptors [19]. Growing interest in KYA centers around its possible involvement in several neurologic diseases with proposed excitotoxic etiologies. as well as its potential involvement in the regulation of excitatory synaptic transmission mediated by endogenous EAAs [12]. Guilarte and Wagner reported that increased concentrations of endogenously produced 3-OHKY were present in neonatal rat brain perinatally deprived of vitamin B6 [20]. These authors suggested that the increased levels of 3-OHKY may be responsible, at least in part, for the seizures observed in these animals. It is known that certain forms of seizures in infancy have a firm relationship to vitamin B 6 and successful high-dose pyridoxine treatment for infantile spasms has been reported [21].

Clinical data of patients with infantile spasms

Patient Number

Age at Diagnosis (mos)

EEG

l 2 3 4 5 6 7 8

5 6 6 7 7 9 10 I1

Hypsarrhythmia Hypsarrhythmia Hypsarrhythmia Hypsarrhythmia Hypsarrhythmia Hypsarrhythmia Hypsarrhythmia Hypsarrhythmia

l0

NAD

PEDIATRIC NEUROLOGY

Vol. 10 No. 1

Other Neurologic Diagnosis

Medication at CSF Collection

Tuberous sclerosis Tuberous sclerosis Perinatal asphyxia Perinatal asphyxia Tuberous sclerosis Perinatal asphyxia Tuberous sclerosis Developmental delay (unknown etiology)

Phenobarbital Sodium Valproate Phenobarbital Sodium valproate Sodium valproate, nitrazepam Phenobarbital Sodium valproate None

Table 2.

E ,5=1.0

A

>-

A

0.8 A AA

~h ~O.G

g

A ZL

~0.4

Con'trol (N =10)

I 'S (N=8)

Figure 2. CSF 3-OHKY levels in controls and patients with infantile spasms before specific treatment.

Johnson and Ascher initially reported that glycine potentiates NMDA electrophysiologic responses and proposed the existence of an allosteric modulator site on the NMDA receptor complex which is sensitive to glycine [22]. Several NMDA receptor antagonists, including KYA, have been demonstrated to act at the glycine allosteric site on the NMDA receptor complex. Accumulation of QUIN, an NMDA receptor agonist, and neuroactive kynurenines are of potential significance in human neurologic diseases because of their neurotoxic and convulsant properties [23]. A central controversy in the study of neurotransmitter levels in neurologic disease is whether abnormal concentrations are the cause or merely an effect of a given disorder. Although abnormally low concentrations of KYA and high concentrations of 3-OHKY were found in patients with infantile spasms, and these compounds have known neuroactive properties, the abnormal kynurenine concentrations found in these patients may represent a response by certain neurons to some other type of injury. Although significant differences in the levels of KYA and 3-OHKY occurred in the CSF of infantile spasm pa-

E 1.2. "-" 1.0< >-

0.8-

8

~= 0.6 eQ)

o

Infantile Spasms (n = 8)

3-Hydroxykynurenine Kynurenic acid

0.19 -+ 0.08 0.78 -+ 0.21

0.73 - 0.18' 0.17 +- 0.09*

t 0% °o

h (/3

cO 0

Compound

Controls (n = 10)

* Values are mean - S.D. (ng/ml CSF) * Significantly different from controls P < .05.

oo

g 0.2

CSF kynurenine metaboHte levels in infantile spasms*

0.4

0.2 AA A

C)

Control (N =10)

I' S (N=8)

Figure 3. CSF KYA levels in controls and patients with infantile spasms before specific treatment.

tients compared to age-matched controls, the small number of patients in this study necessitates further investigation of the relationship between seizures and CSF levels of TRP metabolites. Of considerable interest would be measurement of other metabolites with known neuroactive properties, such as QUIN, in the CSF of seizure patients. References [1] Jeavons PM, Bower BD, Dimitrakound M. Long term prognosis of 150 cases of West syndrome. Epilepsia 1973;14:153-64. [2] Woert MH, Rosenbaum D, Howieson J, Bowers MB. Long term therapy of myoclonus and other neurologic disorders with L-5hydroxytryptophan and carbidopa. N Engl J Med 1977;296:70-5. [3] Hrachovy RA, Frost JD, Kellaway P. Sleep characteristics in infantile spasms. Neurology 1981;31:688-94. [4] Klawans H, Goetz C, Weiner WJ. 5-Hydroxytryptophan induced myoclonus in guinea pigs and the possible role of serotonin in infantile spasms. Neurology 1973;23:1234-40. [5] Lapin IP. Kynurenines and seizures. Epilepsia 1981;22:257-65. [6] Gal EM, Sherman AD. L-Kynurenine: Its synthesis and possible regulatory function in brain. Neurochem Res 1980;5:223-39. [7] Freese A, Swartz KJ, During MJ, Martin JB. Kynurenine metabolites of tryptophan: Implications for neurologic diseases. Neurology 1990;40:691-5. [8] Swartz KJ, Matson WR, MacGarvey U, Ryan EA, Beal MF. Measurement of kynurenic acid in mammalian brain extracts and cerebrospinal fluid by high-performance liquid chromatography with fluorometric and coulometric electrode array detection. Anal Biochem 1990; 185:363-76. [91 Beai MF, Matson WR, Swartz KJ, Gamache PH, Bird ED. Kynurenine pathway measurement in Huntington's disease striatum: Evidence for reduced formation of kynurenic acid. J Neurochem 1990;55: 1327-39. [10] Moroni F, Lombardi G, Robitaille Y, Etienne P. Senile dementia and Alzheimer's disease: Lack of changes of the cortical content of quinolinic acid. Neurobiol Aging 1986;7:249-53. [11] Stone TW, Cormick JH. Quinolinic acid and other kynurenines in the central nervous system. Neuroscience 1985;15:597-617. [12] Conniek JH, Carla V, Moroni F, Stone TW. Increase in kynurenic acid in Huntington's disease motor cortex. J Neurochem 1989; 52:985-7. [13] Matson WR, Langlais PJ, Volicer L, Gamache PH, Bird ED, Mark KA. n-Electrode three-dimensional liquid chromatography with electrochemical detection for the determination of neurotransmitters. Clin Chem 1984;30:1158-61. [14] Matson WR, Gamache PH, Beal MF, Bird ED. EC array sensor concepts and data. Life Sci 1987;41:905-8. [15] Yamamoto H. Studies on CSF tryptophan metabolism in infantile spasms. Pediatr Neurol 1991;7:411-4. [16] Silverstein F, Johnston MV. Cerebrospinal fluid monoamine metabolites in patient with infantile spasms. Neurology 1984;34:102-5. [17] Schwarcz R, Brush GS, Foster AC, French ED. Seizure activ-

Yamamoto et al: Kynurenic Acid and Spasms

11

ity and lesions after intrahippocampal quinolinic acid injections. Exp Neurol 1984;84:1-17. [18] Ileyes MP, Quearry BJ. Quantification of kynurenic acid in cerebrospinal fluid: Effects of systemic and central L-kynurenine administration. J Chromatogr Biomed 1990;530:108-15. [19] Beal MF, Matson WR, Storey E, et al. Kynurenic acid concentrations are reduced in Huntington's cerebral cortex. J Neurol Sci 1992; 108:80-7. [20] Gnilarte TR, Wagner HN Jr. Increased concentrations of 3-hy-

12 PEDIATRIC NEUROLOGY

Vol. 10 No. 1

droxy kynurenine in vitamin B6 deficient neonatal rat brain. J Neulochem 1987;49:1918-26. [21] Blennow G, Strarck L. High dose B6 treatment in infantile spasms. Neuropediatrics 1986;17:7-10. [22] Johnson JW, Ascher P. Glycine potentiates the NMDA re.. sponse in cultured mouse brain neurons. Nature 1987;325:529-31. [23] Saito K, Nowak TS Jr, Markey SP, Heyes MP. Mechanism of delayed increases in kynurenine pathway metabolism in damaged brain regions following transient cerebral ischemia. J Neurochem 1993;60: 180-92.