A comparative Study of Serine racemase in normal and schizophrenic patients

Dr Fatima Shad Kaneez Professor

Panjwani Center For Molecular Medicine and Drug Research International Center For Chemical and Biological Sciences

University of Karachi. Pakistan

1. Schizophrenia is a brain disease that has afflicted human since the beginning of written history.

2. Several lines of evidence focus on a direct involvement of the glutamergic system in the pathophysiology of psychosis.

3. The hypo function of the ionotropic glutamate N-methyl-D-Aspartate Receptor (NMDAR) has been proposed as a model of schizophrenia in humans.

4. D-serine, an endogenous co agonist to the NMDA subtype glutamate receptor, may be implicated in schizophrenia (SCZ)

5. Differences between the plasma serine racemase levels of normal and schizophrenic patients and estimated the D-isomers excreted in their urine indicating the importance of serine racemase in Schizophrenia.

Present study aims to:

(1) Identify biological factors responsible for alterations in D-serine synthesis

(2) Measure differences between the plasma serine racemase levels of normal and schizophrenic patients

(3) Estimate the D-isomers excreted in their urine. (4) Factors affecting regulation of serine

racemase activities

The "Serine racemase" levels in the plasma of normal and mental health patients were analyzed by gas chromatography (GC) and GC-mass spectrometry techniques.The components in the samples were identified by comparison of their mass spectra to reference data bases in conjunction with their retention indices, and they were quantified as appropriate.Later on the regulation of serine racemase activity by amino acids such as glycine and l-aspartic acid were measured as literature indicated that alterations in the concentrations of these amino acids might play a role in the regulation of d-serine synthesis.

Urine samples of both normal and schizophrenic patients were analyzed by GCMS (Gas Chromatography Mass Selectivity) detector.We are proposing that racemization of serine (which is catalyzed by serine racemase, a pyridoxal 5'-phosphate-dependent enzyme expressed mainly in brain and liver) is associated with Schizophrenia.

Racemization of serine is catalyzed by serine racemase, a pyridoxal 5'-phosphate-dependent enzyme expressed mainly in brain and liver. NMDA receptor over activation has been implicated in a number of pathological conditions and inhibitors of serine racemase are thus potentially interesting targets for therapy

Potentially relevant pyridoxal phosphate-dependent reaction pathways of serine catalyzed by serine racemase

SUBJECTS & METHODS Blood collection: Forty drug naïve patients with

a DSM- 1VR Diagnosis of Schizophrenia (American Psychiatric Association 2000) and attending the Psychiatric Department at the Al Ain Hospital, Al Ain (UAE), were recruited for the study.

Approval was obtained from the Ethics Review Board and Informed consent was obtained from the patients prior to collection of blood and urine samples.

Whole blood (20 ml) was collected from fasted (12 hours) patients. Special attention were applied to ensure the optimization of the serum state for measuring serine racemase.

METHODS

Organic analysis: The "Serine racemase" levels in the plasma of

normal and mental health patients were analyzed by gas chromatography (GC) and GC-mass spectrometry techniques.

The components in the samples were identified by comparison of their mass spectra to reference data bases in conjunction with their retention indices, and were quantified as appropriate.

Blood sample were stored at -70°C for further use. Isolation soluble serum fraction of serine racemase, plasma was re suspended in *lyses buffer Lysed by three 20 s bursts of sonication. After centrifugation (15000×g, 30 min, 4°C), the resulting supernatant will be purified by ion exchange chromatography using Q-Sepharose Fast Flow (pH 7.2, lyses buffer, elution by NaCl gradient) and gel filtration chromatography (lysis buffer+150 mM NaCl).

*(50 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) pH 7.2, 10% (v/v) glycerol, 1 mM MgCl2, 0.1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, 0.05% (w/v) polyethyleneglycol 8000, 50 mM NaCl and 50 M pyridoxal 5'-phosphate (PLP))

Total run time was shortened from 45 to 25 minuteswithout any significant loss of resolution.

Gas Chromatography before optimization

Gas Chromatography after optimization

CLINICAL RELEVANCE

There is increasing evidence from pharmacological (Hashimoto et al 2003) and genetic (De Miranda et al 2000) studies that suggest that D-serine, an endogenous coagonist to the NMDA subtype glutamate receptor, may be implicated in schizophrenia (SCZ) (Yamada et al 2005).

Although an association of genes for D-serine degradation, such as D-amino acid oxidase and G72, has been reported, a role for D-serine in SCZ has been unclear.

We believe that the findings from this study, along with other preclinical and clinical data on dopamine-glutamate interaction will have implications for new pharmacological strategies for the treatment of psychosis in general and SCZ in particular.