tournol ot Newt,( hernistrY Lippincott-Raven Publishers, Philadelphia n 1995 International Society for Neurochemistry y-Glutamylglutamine and Taurine Concentrations Are Decreased in the Cerebrospinal Fluid of Drug-Naive Patients with Schizophrenic Disorders K . Q. Do, *C. J. Lauer, *W. Schreiber, M . Zollinger, U. Gutteck-Amsler, M. Cuénod, and *F. Holsboer Brain Research Institute, University ofZürich, Zürich, Switzerland; and *Max Planck Institute of Psychiatry, Clinical Institute, Munich, Germany Abstract: HPLC and gas chromatography-mass spec- monoaminergic and, in particular, the dopaminergic trometry analyses of 18 amino acids, N-acetylaspartate, systems; whereas the other claims a role for the excit- N-acetylaspartylglutamate, and 5-hydroxyindoleacetic atory amino acids, especially glutamate. acid, derived from serotonin, and homovanillic acid, de- Based on the psychotomimetic properties of amphet- rived from dopamine, were performed in CSF collected amine, which is dopamine agonist, and observations a with either a from group of patients schizophrenia who that almost all antipsychotic medications counteract had been drug free for at least 1 year (n = 5) or were drug naive for psychotropic drugs (n = 21) and in 15 dopamine activity in the brain, Matthysse ( 1973) pro- control subjects . Significant differences were found only posed that schizophrenic illness is a manifestation of for taurine (15% lower in the patients) and isoleucine a hyperdopaminergic state. But this initial view was (7% higher) . A number of unidentified substances were not unequivocally supported by subsequent studies, be- detected, one of which proved to be markedly reduced cause conflicting observations were reported regarding (16%) among the schizophrenic patients. Liquid chroma- homovanillic acid (HVA) concentrations in CSF, tography-mass spectrometry with continuous flow-fast plasma, and postmortem brain tissue of individuals atom bombardment interface allowed us to identify this with schizophrenia (for an overview, see Davis et al ., substance as y-glutamylglutamine. The decreased level 1991) . During the past few years, however, evidence of y-glutamylglutamine may reflect a deficiency in the y- glutamyltransferase system, a system probably involved has been accumulated that schizophrenic disorders in glutamate uptake, or a deficiency in glutamine, an im- may be characterized by both hypodopaminergia in the portant precursor of releasable glutamate. Although glu- mesocortical dopamine neurons and hyperdopaminer- tamate was nonsignificantly reduced in the patients, it gia in the mesolimbic dopamine neurons (Davis et al., was one of the five substances (including -/-glutamylglu- 1991) . D2-like receptors were reported to be elevated tamine) that were necessary for the best discrimination in postmortem brain tissue of schizophrenic patients between the schizophrenic patients and the controls . (Seeman et al., 1990), which supports the hypothesis These findings support the notion that the glutamatergic that dopaminergic neurotransmission in schizophrenia system is affected in schizophrenic disorders . In addition, they underscore the need to apply rigid bioanalytical techniques and use drug-naive patients to gain in-depth Received April 7, 1995 ; final revised manuscript received July 3, information on the pathophysiology of brain disorders 1995 ; accepted July 3, 1995 . such as schizophrenia. Key Words: Amino acids-N- Address correspondence and reprint requests to Dr. K. Q. Do at Brain Research Institute, University of Zürich, August-Forel-Strasse Acetylaspartate-N-Acetylaspartylglutamate-5-Hy- 1, CH-8029 Zurich, Switzerland . droxyindoleacetic acid- Homovanillic acid-Cerebro- Abbreviations used: Abbreviations for amino acids are according spinal fluid-Schizophrenia . to the IUPAC-IUB Commission on Biochemical Nomenclature, ce- J. Neurochem. 65, 2652-2662 (1995) . AAA, a-arninoadipic acid ; ANCOVA, analysis of covariance; CF- FAB, continuous flow-fast atom bombardment ; DSM-111-R, Diag- nostic and Statistical Manual of Mental Disorders, third edition, revised ; FMOC, N-9-fluorenylmethyloxycarbonyl ; FMOC-CI, 9- fluorenylmethylchloroformate ; GC, gas chromatography ; y-Glu- Schizophrenia is a nosological concept comprising Gln, y-glutamylglutamine ; FICA, homocysteic acid ; HCSA, homo- a large variety of signs and symptoms that make a cysteinesulfinic acid ; 5-HIAA, 5-hydroxyindoleacetic acid ; HVA, unifactorial explanation of causality unlikely . In psy- homovanillic acid ; LC-MS, liquid chromatography-mass spectrom- etry; NAA, N-acetylaspartate; NAAG, N-acetylaspartylglutamate; chiatric research, two major theories on the pathophys- NMDA, N-methyl-D-aspartate ; OPA, o-phthalaldehyde; OPA-1`15 .5, iology of this disorder have been developed during OPA derivative of 1`15 .5; PCP, phencyclidine; SCID, Structured the past decades . One assumes a dysfunction of the Clinical Interview for DSM-111-R . 2652 REDUCED y-Glu-Gln AND TAURINE IN SCHIZOPHRENIA 2653 is enhanced through overactive response by D2 recep- MATERIALS AND METHODS tors. This concept has been criticized because clozap- Subjects ine, which has only low affinity for D2 receptors, is Twenty-six inpatients ( 15 women, l l men ; age range, clinically a highly effective neuroleptic. Recently, 21-53 years) with a diagnosis of a schizophreniform or however, the group led by Seeman demonstrated the a schizophrenic disorder according to the Diagnostic and existence of a polymorphic D4 receptor to which clo- Statistical Manual (#'Mental Disorders, third edition, revised zapine binds with an affinity 10 times higher than to (DSM-III-R) (American Psychiatric Association, 1987; any other yet-characterized dopamine receptors (Van schizophreniform disorder, n = 9 ; schizophrenic disorder, Tol et al., 1992) . It is interesting that the same group paranoid subtype, n = 13 ; schizophrenic disorder, disorga- found D4 receptors to be elevated in the brain of pa- nized subtype, n = 4), participated in the study . The diagno- tients with schizophrenia (Seeman et al ., 1993) . ses were verified by the Structured Clinical Interview for DSM-III-R (SCID; German version : Wittchen et al., 1990) . The second major hypothesis, as originally formu- None of the participants fulfilled DSM-111-R diagnostic crite- lated by Kim and colleagues (1980), claimed a role ria for any other psychiatric disorders. According to the crite- for excitatory amino acids, i .e., glutamate, in the patho- ria established by the SCID, 15 patients were rated to be physiology of schizophrenia. On the basis of decreased moderately ill and the remaining 11 patients severely ill . levels of glutamate in the CSF of individuals with Four patients had been free of psychoactive medication for schizophrenia, these authors postulated that either a at least I year and one patient for 8 years. The remaining hyperactivity of dopaminergic neurons leads to an en- 21 patients were drug naive in this regard . Furthermore, none of the patients had received any medication during the hanced inhibition of glutamate release or a hypofunc- 2 months before lumbar puncture . tion of the glutamate receptor causes the decreased Fifteen age- and sex-matched subjects (nine women, six glutamate release. Indeed, in rodents, the release of men; age range, 19-58 years) who had been admitted to glutamate from the striatal terminals is inhibited by the neurological ward for diagnostic clarification of health dopamine (see Carlsson and Carlsson, 1990) . The re- problems [headache, hypertension, PRIND (prolonged re- formulation of the glutamatergic hypothesis is based versible ischemic neurologic deficit) occasional spasms, low on the observation that phencyclidine (PCP) elicits back pain] served as the control group. In these subjects, both productive and deficit symptoms of schizophre- internal, neurological, and psychiatric disorders were ex- nia. Although PCP exerts its effects in part via central cluded after a thorough examination. None of these subjects had received any medication during the 2 months before dopamine systems, it is a potent and noncompetitive lumbar puncture . antagonist at the N-methyl-D-aspartate (NMDA) re- The experimental protocol was approved by the Ethics ceptor that has distinct recognition sites for glutamate, Committee for Human Experiments of the Max Planck Insti- aspartate, and glycine. When PCP binds to its receptor tute of Psychiatry . sites within the NMDA ion channels, these voltage- dependent channels are blocked. As a result, the flow CSF sampling procedure of ions into the channel is blocked and the effects After 1 night of bed rest and fasting, subjects were placed in a sitting position at 0800 h. After administration of an of' NMDA agonists such as glutamate or glycine are intradermal xylocaine anesthesia, an 18-G 32, or a 19-G 3'-, antagonized (see Heresco-Levy et al., 1993) . Thus, Yale spinal needle was inserted into the lumbar subarachnoid the current hypoglutamatergic theory of schizophrenia space through the L3-L4 or L4-L5 interspace. After collec- proposes diminished glutamatergic neurotransmission tion of CSF, 5 ml of the fluid was transferred into an ice- at the level of the NMDA-receptor site (Deutsch et cooled test tube. The CSF was vortex mixed and aliquoted al ., 1989 ; Squires and Saederup, 1991) . into five 1-ml vials. These vials were then immediately fro- Although both the dopaminergic and glutamatergic zen and stored at -80°C until analysis . hypotheses of schizophrenia have stimulated intense Materials work