Free amino acid level determinations in normal and schizophrenic brain

Pmg. Neum-Psychophormocol. L+Biol. Psychiot. Printed in Great Britain. AIL rights reserved

027~584tYgQ $0.00 + SO Copyright @ 1989 Pergamon Press plc

lQg9. Vol. 13, pp. 119-126

FREE AMINO ACID LEVEL DETERMINATIONS AND SCHIZOPHRENIC BRAIN FATMA Z.KUTAYl,

$AKlRE

Ege University,

POGDN*,

NURAN i.HARtRt*,

IN NORMAL

GSNDL PEKER* and SERMET

S hool of Medicine, Departments of 'Biochemistry 3 Physiology, Bornova, fzmir, Turkey

(Final form August

ERLACfNl and

1988)

Abstract I(utay Faba Z.,$akire P8gDn, Nuran 1. Hariri, GiinDl Peker and Sermet Erlacin : Free amino acid 7eve1 determinations in normal and schizophrenic brain. Prog. Neuro-Psychopharmacol. and Biol. Psychiat. 1989, 13: jig-126

1. Some disturbances in brain amino acids are reported with regard to pathological changes in schizophrenia: a reduction in GABA content and a reduced activity at some glutamatergic synapses. 2. Comparison of post-mortem brain tissue from control subjects and schizophrenic patients can provide evidence for amino acid alterations in disease. 3. The present study was undertaken to measure free amino acid concentrations in 20 brain regions obtained at autopsy, from normal persons and schizophrenics. Amino acids were extracted, esterified and separated by gas chromatography. 4. The distribution and levels of amino acids in normal persons is in accordance with similar values reported in human post-mortem brain samples by other investigators. 5. The differences in amino acids found in schizophrenic brain samples support the view of disturbed neurotransmission especially with regard to GABAergic and glutamatergic systems in schizophrenia and suggest the possible involvement of other amiro acids as well.

: Amino acid levels, post-mortem

Keywords

human brain, schizophrenia.

Abbreviations: alanine (Ala), asparagine (As NH ), aspartate (Asp), ethylene glycol adipate (EGA), gamma-aminobutyric acid (GABA P , Glutamate (Glu), glutamic acid decarboxylase (GAD), glutamine (GluNH ), glycine (Gly), isoleucine (ILe), leucine (Leu), lysine (Lys), methionine (Met), 8rnithine (Orn), phenylalanine (Phe), proline(Pro), valine (Val).

Introduction Amino acids have started of synaptic

function

neurotransmitters

putative

of dopamine.

types of schizophrenia interaction tic terminal tissue effects

or candidate.

may be a result of a defect

the effects

as neurotransmitters partially

or as neuromodulators

in an effort

to find the "missing"

in brain. Today, while some are proven to be neurotransmitters,

still are considered zophrenia

to gain recognition

during the past decades,

GABA ergic deficiency

receptors

the dopaminergic

may be a characteristic

system

release

observed

others in schi-

systems modulating finding

in some

from the nerve ending

and

, some of the GABA is taken back to the presynap-

while the rest is catabolized butyrate

hyperactivity

in one or more neurotransmission

(Perry et al., 1979). Following

with postsynaptic

to gamma hydroxy

Dopaminergic

enzymatically

in the nerve terminals

(McGeer et al., 1979; McGeer (Cash et al., 1981).

119

and McGeer,

The co-existance

or glial

1981) which

of GABA with

120

F. 2. Kutay etal.

serotonin

was observed

transmission

systems

in some neurons

since human brain is inaccessible

levels during a disease

Regarding

following

to CSF or plasma

to reflect

the putative

information

the relative

on absolute

differences

between

pathologyof schizophrinia,

neurotransmitter

in their brain distribution,

free amino acids in 20 regions

methionine,

the majority

et al., 1985;

group.

the role of amino acids in the etiological

and schizophrenic

(Bjerkensted

amino acid levels may not provide

the first place, but considering and the variability

disorders,

for direct amino acid level determinations,

life, but they may be assumed

and a control

the two neuro-

appear likely in many neuropsychiatric

in the field were restricted

Enna et al., 1977). Post-mortem

between

et al., 1981).

(Nanopoulos

Even though amino acid alterations

of the studies

and there is an interaction

patients:

phenylalanine,

alanine,

of post-mortem

valine,

aspartate,

the authors

glycine,

glutamate,

GABA has

roles of other amino acids determined

the levels of the

human brains in normal subjects

isoleucine,

ornithine,

leucine,

proline,

GABA,

lysine.

Methods Human Post-mortem

Brain Specimens

The brains of schizophrenic normal

persons without

University

Hospital)

patients

who died in Manisa,

any neuropsychiatric

were obtained

disease

at autopsy

(20 regions),

1 ml of 2.5 mM norleucine

was added as the internal

were studied;

time between

(Dept. of Forensic

and examined

The brains were dissected

weighed,

renics whose mean age was 40 and 3 male,

State Psychiatric

standard.

1 female normal

Medicine,

to exclude

put in saturated

Hospital,

and

Ege

any gross pathology.

picric acid (l/10)

and

5 Male, 2 female schizoph-

persons whose mean age was 31

death and autopsy was 65 (48-83) hours for the schizophrrnics

and 48 (20-56) hours for the normal persons. Preparation

of Tissue

for Gas Chromatoqraphic

Tissue was homogenized

at 1400 rpm using a glass-teflon

at 3000 x g for 15 minutes. x 7 cm) containing was collected lyophilized.

The supernatant

7N NH40H through

The lyophilizate

ration

homogenizer

through

under nitrogen

(0.9 cm

The amino acid fraction

in O.IN HCI and transferred

lined screw caps. Esterification

3N HCI, and acetylation

and centrifuged

glass columns

the column with a flow rate of 3 ml/min.

was dissolved

rycle.. The amino acid trifluoro dryness

was eluted

Dowex 50 W x 12 and washed with d.d. water.

by passing

tubes with teflon n.Butanol

Separation

in 5 minutes

acetyl n-butyl

occurred

in 15 minutes

at 100°C with

at 15O'C with trifluoroacetic derivatives

and stored at -2O'C in chloroform

obtained,

and

to acetylation

acid anhyd-

were brought

until gas chromatographic

to sepa-

(Kutay et al., 1983).

Amino Acid Analysis Pye-Unicam

104 gas chromatograph

in 2 nn~!x 180 cm glass columns program

between

60-21O'C.

was used for separation.

containing

The amino acids were separated

0.65 % EGA chromosorb

W.NAW using a 6'C/min,

The areas under the amino acid peaks were calculated

using an

Amino acids in normal and scbizopbrenic brain

121

integrator (Spectra Physics Minigrator) and the actual amino acid levels were determined (Kutay et al., 1983). Results The following amino acids were separated chromatographically : alanine, valine, glycine, isoleucine, leucine, proline, GABA, methionine, phenylalanine, aspartate, glutamate, ornithine, lysine.Since glutamine and asparagine are converted to their acid forms during esterification, they were evaluated together with glutamate and aspartate. The peak of the internal standard, norleucine, was between leucine and proline. The free amino acid distribution in 20 regions of normal, post-mortem human brain is seen in Table 1. The amino acids glycine, GABA, aspartate and glutamate reveal a local distribution pattern which is in accordance with their physiological functions. There is also a striking difference in the distribution of the amino acids alanine, leucine, proline, methionine, phenylalanine,ornithine and lysine. The differences mentioned were also observed when each case was evaluated separately. The free amino acid distribution in post-mortem schizophrenic brain specimens is seen in Table 2. The differences between the two groups are summarized in Table 3. Discussion The majority of amino acids are higher in autopsied brain specimens compared to biopsies (Perry et al., 1971a and b). The continuing activity of some synthetic enzymes, changes in ATP levels and deamination reactions account for these changes which occur during the first few minutes after death (Minard and Mushahwar, 1966; Perry et al.,

1971a; Peterson

and McKean, 1969). Since post-mortem conditions are identical in both groups included in our study, we believe that our results reflect the relative differences which will provide important cues with regard to the possible pathological changes observed in schizophrenia.. Distribution of Amino Acids in Normal Brain In our study, the normal amino acid levels and their distribution is in accordance with the values reported by other investigators (Bachelard, 1981; McGeer et al., 1979; Perry et al., 1971a; Okumuraet al.,

1960) (Table 1.). There is no direct measurement of free

amino acid levels in autopsied schizophrenic brain regions, however there are some studies reporting changes in amino acid levels and related transport systems in the CSF and plasma of schizophrenic patients (Okumura et al., 1960). Changes in Amino Acids in Schizophrenic Brain In our study, in schizophrenics,we found high levels of neutral amino acids in many brain regions (Table 2) compared to normals, which can be attributed to a defect in the L-transport system. High levels of phenylalanine in the thalamus and some cortical areas of schizophrenics (Tables 2 and 3) may imply a defect in the conversion of phenylalanine to tyrosine which will subsequently effect catecholamine metabolism as suggested by

0.50

1.81

1.09

2.87

Dorsal

Subs. nigra 3.05 2.14

3.03 2.98

1.25

2.46

Medulla spinalis

Cerebellar

Asp + AspNH2,

**

Glu + GluNH2

wet weight of tissue

pmol/g.

*

cortex

4.91

6.49 1.65

5.39

3.36

2.17

2.57

2.19

Red n.

2.61

4.34

1.17

4.69

Globus pallidus

2.66 2.91

3.01

Putamen

2.92

1.31 1.42

3.11

Caudate n. (body)

2.59

1.60

2.72

2.31

3.21

1.22

2.54

Caudate n. (head)

Extrapyramidal areas

1.20

2.61

Ventral 1.75

Hippocampus

2.79

1.19

Thalamus

1.51

2.20

2.63

3.39

0.80

1.60

2.48

Amygdala

Limbic cortex

Corpus callosum

1.60

1.53

2.95 1.63

0.84

2.11

Inf.temp.gyr.

Sup.temp.gyr.

1.76

1.96

2.91

0.91

1.71

(fis.calc.)

Occip.

2.99

2.65

3.25

1.19

3.54

(assoc)

Occip.

3.01

2.06

3.38

3.65

2.63

2.21

1.10

gyr.

gyr. 0.93

Postcent.

2.41

Precent.

2.50

1.39

1.81

Leu

0.63

Gly

1.53

Val

2.48 1.94 3.71 0.38

0.93 0.65 1.85 0.90

3.94 4.56 4.92 3.06

9.12 0.87 3.13

1.62 1.32

1.36 1.56

0.50

1.01 0.53

0.95

1.04

0.69

0.67

1.59

3.57

1.30

2.63

3.76

1.35

1.11

2.46

3.78

1.27

3.41

2.80

1.38

2.84

1.40 0.83

1.04

2.45

GABA

0.51 0.31 1.13 0.55

0.51 0.59 0.50 0.40

1.73 0.85 0.48

1.33 1.06

0.66

0.56

0.89

0.57 0.57

3.20

5.41

12.04

3.30

4.61

3.25

4.16 0.51

3.32

0.55

3.26

3.08

2.42

4.06

2.00

4.76

2.83

3.93

4.87

3.64

3.70

2.90

** Asp

0.72

0.88

0.56

0.61

0.46

0.41

0.28

0.50

0.88

1.28

0.62

0.66

0.62

0.91

0.86

0.59

0.61

0.44

Phe

(Normal)*

0.36

Met

of Human Brain

0.68

Pro

Regions

1

1.48

0.56

0.40

0.56

0.63

0.52

0.38

0.89

0.45

0.46

0.82

0.50

0.53

0.31

ILe

in Different

Cerebral hemispheres Frontal pole

Ala

Free Amino Acids

Table

0.29 -

9.12 13.09 6.49

0.47 0.29

6.78 9.56

0.75

-

15.5

0.34

7.61

9.68

0.58

0.31

6.56

0.24

8.06

0.32

3.76

10.8

0.14

9.08

13.53

0.25 0.08

7.28

0.32

9.81 8.69

0.15 0.10

7.25 8.83

0.09

Orn

10.72

** Glu

1.05

1.57

3.49

1.45

2.17

1.51

1.48

1.93

1.03

0.32

1.06

2.27

0.42

1.03

0.60

0.52

1.23

0.74

1.15

0.59

Lys

2.86

2.87

Ventral

Dorsal

0.90

1.38

Putamen

Globus pallidus

0.99

Cerebellar

cortex

2.62

Medulla spinalis

Red n.

1.52

0.148

Caudate n. (head)

Caudate n. (body)

areas

1.91

Extrapyramidal

1.09

Hippocampus

1.64

Amygdala

Limbic cortex

Corpus callosum

1.86

Sup.temp.gyr.

2.01

2.45

(fis talc.)

Occip.

1.92

Inf.temp.gyr.

(assoc)

Occip.

1.45

1.56

gyr.

gyr.

Precent.

Postcent.

1.56

Cerebral hemispheres Frontal pole

Ala

1.25

1.65

2.00

1.17

1.42

1.31

1.22

1.09

1.20

1.19

0.80

0.50

1.53

0.84

0.91

1.19

0.93

1.10

0.63

Val

2.38

3.09

3.12

4.58

2.07

0.68

1.02

2.89

4.29

2.01

1.75

1.95

2.33

1.95

2.51

1.58

2.07

1.71

2.12

Gly

Free Amino Acids

2.46

2.62

1.65

1.87

1.04

1.65

1.69

1.99

2.04

1.74

1.31

1.35

1.72

1.62

2.48

1.71

1.45

1.69

1.72

Leu

0.48

0.90

0.90

3.80

0.59

0.37

0.44

0.72

1.06

3.89

0.76

0.76

0.42

0.46

0.45

0.54

0.81

0.44

1.36

1.16

2.32

1.20

0.70

0.87

0.59

0.69

0.66

0.87

0.80

0.82

0.84

1.14

1.14

0.80

0.80

1.01

Pro

Regions

0.55

ILe

in Different

Table 2

1.69

0.85

4.67

5.14

4.88

2.17

2.64

1.06

1.72

1.36

1.33

0.49

1.94

1.63

2.24

1.30

1.96

1.53

1.45

GABA

0.39

0.72

0.47

0.54

0.28

0.50

-

-

-

0.51

0.39

0.21

0.43

0.64

0.31

0.52

0.34

0.45

Met

0.56

0.76

0.88

0.71

0.68

0.73

0.98

1.01

0.49

0.57

0.42

0.53

0.95

0.65

0.69

0.48

0.55

0.64

Phe

2.26

1.65

4.73

2.18

1.35

3.02

2.65

3.71

1.80

2.75

1.73

1.68

1.98

3.27

2.75

2.28

2.41

2.49

Asp

of Human Brain (Schizophrenic)

13.41

7.49

20.05

21.68

18.32

17.0

18.72

15.39

10.14

14.27

7.63

14.92

14.85

16.66

6.66

12.48

11.72

17.81

Glu

0.32

0.45

1.03

0.64

0.40

0.29

0.39

0.029

0.089

0.21

0.37

0.23

0.13

0.21

0.20

0.29

0.17

0.18

0.21

Orn

1.53

1.13

2.20

0.96

1.20

0.93

1.19

0.26

0.51

0.85

0.72

0.84

0.85

1.27

0.86

0.92

0.34

1.09

Lys

124

F. Z. Kutay et al.

c c -4 -

4

-

I

P -

.

- 4

-4

Amino acids in normal and schizophrenic

brain

125

Potkin (Potkin et al., 1983). Table 3 shows a fall in GABA levels in most of the regions in schizophrenic brains; this finding supports other investigators'opinions regarding a deficiency in GABA ergic activity in the etiological pathology of schizophrenia (Bracha and Kleinman, 1986; Ko et al., 1985; Perry et al., 1979). In schizophrenics,glutamate levels show a negative correlation with GABA in our study (Table 3), which may imply a decline in GAD activity in schizophrenia;a possible neurotoxic effect of glutamate may also be contributing to the symptomatic manifestions of schizophrenia. Evaluation of our results also reveal changes of some amino acids without known neuroactive functions, in some regions of schizophrenic brains (Tables 2 and 3). Another finding is a change in the distribution of some amino acids in schizophrenia (Tables 1 and 2). Even though these changes reveal possible roles of amino acids in the schizophrenic syndrome, we don't have enough informationyet to give explanations for all the differences observed. Conclusion The comparison of free amino acid levels in normal and schizophrenic post-mortem brain samples show that there is disturbed neurotransmissionespecially with regard to GABA ergic and glutamatergic systems in schizophrenic brains. Our data also suggest the possible involvement of other amino acids. Further research involving post-mortem studies will elucidate the roles of amino acids in health and disease. Acknowledgement This study was supported by the Ege University Research Foundation Grant No:025/84. References BACHELARD,H.S. Biochemistry of centrally active amino acids. (1981) In: Amino Acid Neurotransmitters,F.V.De Feudis and P.Mandel (Eds.) pp: 475-497, Raven Press, NewYork. BJERKENSTED,L., EDMAN,G., HAGENFELDT,L., SEDVALL,G., WIESEL,F.A. (1985) Plasma amino acids in relation to cerebrospinal fluid monoamine metabolites in schizophrenic patients andhealthy controls. Brit.J.psychiat.147, 276-282. BRACHA,H.S. and KLEIMAN,J.E. (1986) Post-mortem neurochemistry in schizophrenia. Psychiat. Clin.Nerth America. 9 (1): 133-141. CASH,C.D.,RUMIGNY,J.E.,MANDEL.P.,MAITRE,M.(1981) Enzymology of the alternative reductive pathway of GABA catabolism leading to the biosynthesis of gama hydroxy butyrate. pp: 527-535, Raven Press, NewYork. ,.CNNA,S.J., WOOD,J.H., SNYDER,S.H. (1977) Gamma-amino butyric acid (GABA) in human cerebrospinal fluid. Radioreceptor assay.J.Neurochem.-28: 1120-1124. KO,G.N., KORPI,E.R.,FREED,M.J.,ZALCHMAN,S.J., BtGELOW,L.B., 1985. Effect of valproic acid on behavior and plasma amino acid contentrations in chronic schizophrenic patients. Biol. Psychiatry -20: 209-215. KUTAY.F., 0NAT.T.. ERLACIN,S. (1983) Plasma, kas ve karacigerde serbest amino asidlerin gaz-likid kromatografisi ile analizi. E.D.T.F. dergisi, 22(4): 1007-16. MC GEER,P.L., ECCLES,J.C., MC GEER,E.G. (1979) Molecular Neurobiology of the Manzaalian Brain. pp: 199-230, Plenum Press, NewYork and London.

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MC GEER,P.L. and MC GEER, E.G. (1981) Amino acid neurotrasmitters. In: Basic Neuro-, chemistry,Siegel, G.J., Albers, R.W., Agranoff, B.W., Katzman, R. (Eds.) pp: 233-253, Little, Brown and company, Boston. MINARD,F.N. and MUSHAHVAR,L.K. (1966) Synthesis of gana butyric acid from a pool of glutamic acid in brain after decapitation. Life Sciences, 5: 1409-1413. NANOPOULOS,D., MAITRE,M., BELIN,M.F., AGUERO,M., PUJOL,J.F., GAMRANI,H., CALAS,A. (1981) Autoradiographic and immune cytochemical evidence for the existence of GABA-ergic neurons in the nucleus raphe dorsalis-possible existence of neurons containing 5HT lutamate decarboxylase. In: Amino Acid Neurotransmitters, F.V. De Feudis and P.Mantel 9 Eds.) pp: 519-525, Raven Press, NewYork. OKUMURA,N., OTSUKI,S., KAMEYAMA,A. J.Biochem. 47(3): 315-320.

(1960) Studies

on free amino acids

in human brain.

PERRY,T.L., BERRY.K., HANSEN,S., DIAMOND,S., MOK,C. (1971a) Regional distribution amino acids in human brain obtained at autopsy. J.Neurochem. -18: 513-519.

of

PERRY,T.L.,HANSEN,S., BERRY,K., MOK,C., LESK,D. (1971b) Free amino acids and related compounds in biopsies of human brain. J.Neurochem. -18: 521-528. PERRY,T.L. KISH,S.J., BUCHANON,J. (1979) Garena amino butyric of schizophrenic patients. Lancet, I: 237-239.

acid deficiency

in brain

PETERSON,N.A. and MC KEAN,C.M. (1969) The effects of individual amino acids on the corporation of labelled amino acids into proteins by brain homogenates. J.Neurochem. 16: 1211-1217. POTKIN,S.G., CANNON-SPOOR,H.E., De LISI,L.E., NECKERS,L.M., WYATT,R.J. (1983) Plasma phenylalanine, tyrosine and tryptophan in schizophrenia. Arch-Gen. Psychiatry. 40: 749-752. Inquiries

and reprint

requests

Dr.Fatma Z.KUTAY Assoc.Prof. of Biochemistry Ege University, Medical School Department of Biochemistry Bornova,fzmir TURKEY

should

be addressed

to: