Impairment of glutamatergic system regulation contributes to the development of psychosis in schizophrenia (1, 2, 3). Glutamate concentration and the activity and concentration of glutamate receptors and transporters are altered in the brain of patients with schizophrenia in comparison with mentally healthy persons; besides, strong antagonists of NMDA glutamate receptors induce psychotomimetic effects. These facts serve a basis for the »glutamate hypothesis« of schizophrenia pathogenesis that is prominent today (4). Glutamate neurotransmitter signal transduction depends on amounts and activity of glutamate receptors and transporters, as well as on activity of glutamate converting enzymes. Glutamate concentration in tripartite synapses is determined by equilibrium between its transport, binding by specific receptors, and metabolism by neuronal and glial enzymes, and the concentration can change due to impairment of glutamate metabolism leading to neurological or mental pathologies, including psychoses (5, 6, 7). As a result of investigations of human autopsy brain, characteristic features of glutamate metabolism in schizophrenia have been revealed in comparison with mentally healthy individuals. These features consist in changing both the concentration and intracellular localization of key glutamate metabolizing enzymes such as glutamine synthetase, glutamine synthetase-like protein, glutamate dehydrogenase isoenzymes (GDH), and regulatory links that control the ratio of these enzymes (8). Neurochemical studies of the brain are important for understanding the pathogenesis of schizophrenia, but the search for biochemical changes in the blood of schizophrenic patients is relevant and important in revealing biomarkers helping to choose the right antipsychotic therapy and predict its efficacy (9). There is a need for an inexpensive and reliable pathogenesis-related biomarker for prediction of antipsychotic therapy efficacy in patients with schizophrenia. From this point of view, blood platelets deserve special attention because they possess glutamate system components such as glutamate receptors, transporters (glutamate-dependent ion channels) (10), and some glutamate metabolizing enzymes (11, 12).
The key glutamate metabolizing enzyme, glutamate dehydrogenase (GDH, EC 22.214.171.124), catalyzes the reaction:
Three isoforms of GDH (GDHI, GDHII, and GDHIII) have been described in the human brain, differing in their strength of association with the cell membrane (12, 13, 14, 15). Using antibodies to the human brain GDH isoforms, two isoenzymes of GDH, similar to GDHI and GDHII, are found in extracts of human platelets, whereas the amount of membrane-associated GDHIII isoform is below the threshold of immunoblotting detection (11). The presence of GDH in platelets is confirmed not only by immunoblotting, but also by the presence of its enzymatic activity (16, 17).
In this study, we evaluated the relationship between the activity of platelet GDH and the efficacy of antipsychotic pharmacotherapy in a group of patients with chronic acute schizophrenia.
Materials and Methods
A group of patients with paranoid schizophrenia (F20.0 according to ICD-10) with chronic course of the disease, all inpatients due to their illness (n = 50: 47 men and 3 women aged 25–56 years, median 34 years), was examined in initially acute psychotic state (hallucinatory paranoid or paranoid syndrome), before and after a treatment course with typical and atypical antipsychotics. A total of 50 patients entered the study, and for 45 patients the study was completed. Exclusion criteria were organic central nervous system diseases and acute and chronic somatic diseases.
Informed consent was obtained from all the participants of the study, the study was performed in conformance with the Declaration of Helsinki ethical guidelines, and ethical approval for the study was obtained from the Ethics Committee of the Mental Health Research Centre (Protocol N343, 14.04.2017). The patients were assessed twice by the attending psychiatrist, before and after the treatment course, using the Positive and Negative Syndrome Scale (PANSS) (18). In accordance with the peculiarities of the mental state of each patient, psychopharmacotherapy individually selected by clinicians was carried out using typical or atypical antipsychotics. The end of the treatment course was determined as improvement in the patient’s condition sufficient for discharge from the hospital (about two months after the initiation of the therapy).
When assessing patients’ state using PANSS, »responder« category for each PANSS subscale (PANSSpositive subscale, PANSSnegative subscale, and PANSS subscale of general psychopathology) was assigned to those patients who had not less than 30% reduction of the corresponding PANSS subscale score. Table I contains data on the patients.
Patient Demography and Clinical Data.
|Gender||47m / 3f|
|Education: secondary/secondary incomplete special/higher/higher (number of patients)||14 / 17 / 9 / 10|
|Hereditary burden (+/–)||11 / 39|
|Predominant psychopathology: hallucinatory/disorganization delusional/ (number of patients)||21 / 21 / 8|
|Hospitalizations: 1/2/3 (number of patients)||5 / 8 / 37|
|Antipsychotic therapy: atypical/typical/mixed (number of patients)||19 / 19 / 12|
|Median; minimum – maximum||25-, 75% – quartiles|
|Age (years)||34; 20–56||28; 42|
|Disease duration (months)||96; 2–420||48; 240|
|Age of manifestation (years)||25; 17–42||19; 28|
|PANSS positive at baseline||23; 10–35||21; 28|
|PANSS negative at baseline||26; 12–39||21; 29|
|PANSS psychotic at baseline||47; 25–71||43; 53|
|PANSS total at baseline||96; 58–141||89; 106|
Blood samples were collected after overnight fasting into evacuated tubes containing anticoagulant agent (3.2% Na-citrate buffer).
Each blood sample was treated individually within 2 hours after blood collection. Initially, the whole blood was centrifuged for 15 min at 200 g and 20 °C. The supernatant (platelet-rich plasma) was used for the subsequent isolation of platelets.
The platelet-rich plasma was centrifuged for 20 min at 2000 g at 4 °C. The precipitate was resuspended in 0.105 mol/L Na-citrate buffer with 0.105 mol/L glucose (pH 5.7) and centrifuged again for 20 min at 2000 g at 4 °C. The platelet precipitate was resuspended in 0.05 mol/L Tris-HCl buffer, pH 7.0, with 50% glycerol, and stored at –20 °C.
Immediately before determining the activity of GDH, 50 mmol/L K-phosphate buffer pH 7.4 containing dodecyl-β-D-maltoside was added to the platelet sample to final concentration of 1%. Lysis was carried out for 10 min at 25 °C, then the samples were centrifuged for 10 min at 4000 g at 4 °C. The resulting supernatant was diluted 5-fold in 50 mmol/L K-phosphate buffer, pH 7.4 (16).
GDH activity measurement
The activity of GDH was determined by a spectrophotometric method (19) with modifications (17). The sample volume added to 400 µL of the reaction mixture was 125 µL. The reaction mixture composition was 0.1 mmol/L NADH, 0.1 mol/L NH4Cl, 10 mmol/L α-ketoglutarate, 1 mmol/L ADP, and 2.6 mmol/L EDTA in 0.01 mol/L Tris-HCl pH 8.0 buffer. The measurement of the rate of reaction (1), in this case directed to the right, is based on the loss of absorption of NADH recorded by spectrophotometry at 340 nm for 3 min. To calculate the enzymatic activity, the value of the molar extinction coefficient of NADH, 6.22×10-3 l×mol-1 ×cm-1 was taken, and the specific activity of GDH per 1 mg of protein is given in Figures 1 and 2.
Protein concentration was determined by the Lowry spectrophotometric method using a Bio-Rad DC Protein Assay Kit (USA) in accordance with the protocol and using bovine serum albumin (Sigma-Aldrich) as a protein standard for calibration.
The significance of differences between experimental groups was determined using »nonparametric analysis«. The significance of differences, changes in the parameters, and connections between them were assessed using the Mann–Whitney U-test, Chi-square test with Yates correction, Spearman correlation coefficients determination, and the Wilcoxon matched pair test. Differences and correlations were considered significant at p<0.05.
The schizophrenia-patient and control groups were matched in age and gender, allowing between-group comparison of GDH activity. Significantly reduced GDH activity was found in the patient group both before and after the treatment course compared with the control group (Mann–Whitney U-test, p<0.01 and p<0.04, respectively), thus we reproduced our previous data (17).
The results of GDH activity determination for the schizophrenia patients before (baseline) and after the treatment course are shown in Figure 1.
When regarding the individual values of GDH activity in the patient group before the treatment course, substantial variation in values was noted. Therefore, this group of patients was divided into two subgroups by the median of the baseline (initial) GDH activity for further statistical analysis. The Wilcoxon matched-pair test showed that the GDH activity significantly increased after the treatment (p = 0.002) in the subgroup with the activity of baseline GDH below the median. Figure 2 shows obvious dynamics of GDH activity during the treatment in this subgroup: the majority (18 patients) demonstrated an increase, and the other 5 showed no change or a decrease in GDH activity, whereas no significant changes in GDH activity were observed after the treatment in the subgroup with the baseline GDH activity above the median.
After the treatment course, significant correlations of GDH activity levels with PANSS negative and PANSS total score were revealed in the subgroup with the baseline GDH activity above the median (Spearman correlation coefficients R = 0.56, p = 0.007 and R = 0.45, p = 0.04, respectively). Besides, there was a significantly higher number of responders in this subgroup (for instance, by PANSS positive subscale, Chi-square with Yates correction = 9.1, p = 0.001) (Table II).
Distribution of Responders and Non-responders (in Percentages of Total Group) by PANSS between the Subgroups of Patients with Baseline GDH Activity Below and Above the Median.
|PANSS subscale||Below median GDH||Above median GDH|
|General psycho- pathology||7||42||18||33|
On the other hand, there was a significantly higher number of non-responders in the subgroup with baseline GDH activity lower than the median. Among these were non-responders by PANSS negative subscale score (Chi-square with Yates correction = 7.5, p = 0.006), by PANSS psychotic (Chi-square with Yates correction = 5.7, p = 0.02), and by PANSS total score (Chi-square with Yates correction = 3.8, p = 0.04).
As we indicated in the Introduction, we previously found two GDH forms (similar to GDHI and GDHII) in human platelet extracts (9, 10, 11) when the proteins were extracted using detergent (sodium dodecylsulfate, SDS) and the samples boiled with SDS according with the ECL-immunoblotting protocol developed for evaluation of GDHI and GDHII in human brain extracts using the same immunoprobes (i.e. antibodies recognizing human brain GDHI and GDHII) (8). When estimating GDH amount by immunoblotting, the sum of GDHI and GDHII forms was taken into consideration, and no significant difference was found between the group of patients with paranoid schizophrenia and controls (11).
In the present work, a soft extraction method was employed for GDH enzymatic activity measurement using n-dodecyl β-D-maltoside, which preserves the enzymatic activity. The GDH activity was found to be significantly higher in the control group than in the patient group. Obviously, SDS and n-dodecyl β-D-maltoside may differently extract the GDH forms from platelets, causing different dissociation of the GDH forms particularly from platelet mitochondria.
We would like to emphasize that the method of GDH enzymatic activity determination is more easily standardized and is not so expensive and time consuming in comparison with ECL-Western immunoblotting, and thus the activity determination is preferable for routine clinical analyses.
Generally, only in the subgroup of patients with baseline GDH activity below the median was the GDH activity significantly increased after the treatment, and a significantly higher number of non-responders to antipsychotic treatment was found in this subgroup.
Correspondingly, a significantly higher number of responders was found in the other subgroup with baseline GDH activity above the median, and significant correlations of GDH activity levels with PANSS negative scores and PANSS total scores were revealed after the treatment in this subgroup.
Significantly different uneven distribution of responders and non-responders between two subgroups of patients with paranoid schizophrenia (with baseline GDH activity above and below the GDH median) indicates that measurement of platelet GDH activity in patients with paranoid schizophrenia before the course of antipsychotic treatment might be useful for individual prediction of the efficacy of antipsychotic pharmacotherapy.
Positive and Negative Syndrome Scale
We thank Richard H. Lozier for his very useful comments and assistance in work on this manuscript.
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