Membrane function alterations in erythrocytes from mood disorder patients

RESEARCH

SA Psych Rev 2003;6:11-20

Membrane function alterations in erythrocytes from mood disorder patients a

Subhash D Katewa b Kunjan R Dave c S

Author Barnard McDonald

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RESEARCH

SA Psych Rev 2003;6:11-20

Membrane function alterations in erythrocytes from mood disorder patients a

Subhash D Katewa b Kunjan R Dave c Surendra S Katyare a Department of Biochemistry, State University of Buffalo, Buffalo, NY, USA c Department of Biochemistry, Faculty of Science, M.S.University of Baroda, Vadodara, India b Department of Neurology (D 4-5), School of Medicine, University of Miami FL USA

ABSTRACT Objectives: To examine erythrocyte membrane functions in mood disorder patients and to establish possible diagnostic marker parameter(s). Design: Collection of blood samples from mood disorder patients and age-matched control volunteers.Preparation of erythrocyte membranes for the proposed studies. Setting: Out patients / in patients, psychiatry ward, Civil Hospital, Ahmedabad, Gujarat, India, Department of Biochemistry, Faculty of Science, M.S.University of Baroda, Vadodara, Gujarat, India. Subjects: Unipolar and bipolar subjects. Control subjects (randomly selected volunteers). Results: The most significant results were a duration dependent decrease in the TPL/CHL ratio (mole:mole),changes in both the substrate and temperature kinetics properties of AChE and elevated plasma BChE activity in the mood disorder patients. Conclusion: The results suggest that the altered lipid profiles and the TPL/CHL (mole: mole) ratio and the altered temperature-dependent activity coefficients of erythrocyte membrane AChE and elevated plasma BChE activities could serve as useful diagnostic pointers for mood disorders. Keywords: Membrane function; Erythrocytes; Mood disorder Abbreviations used: ACh - Acetylcholinesterase; ACTI - Acetylthiocholineiodide; BChE - Butyrylcholinesterase; BCTI -Butyrylthiocholineiodide; CHL - Cholesterol; DPH - 1,6-Diphenyl –1,3,5-hexatriene; DTNB - 5,5’ – Dithiobis(2-nitrobenzoic acid); ETPZ.HCl - Ethopropazine hydrochloride; Lyso, Lysophospholipid; PA - Phosphatidic acid; PC - Phosphatidylcholine; PE - Phosphatidylethanolamine; PC Phosphatidylinositol; PS - Phosphatidylserine; SPM - Sphingomyelin; SDS - Sodium dodecyl sulfate; TPL - Total phospholipid.

INTRODUCTION Mood fluctuation is a common and normal component of human behavior and involves both depression and anxiety. However, when these emotional states become uncontrollable, they lead to behavioral disorders known as mood disorders.1 The diagnostic statistical manual IV (DSM – IV) has classified the mood disorder conditions based on the form and frequency of episodes, as well as the duration.1,2 The mood disorders (also known as affective disorders) fall in two broad groups i.e. major depressive disorder and bipolar disorder (Type 1); the two conditions are also commonly known as unipolar and bipolar conditions. Erythrocyte membrane abnormalities such as decreased methylation activity resulting in decreased PC levels, decreased erythrocyte membrane Na+, K+-and Mg2+ ATPase activity, altered intra-erythrocytic cationic concentrations and changes in AChE activity in mood disorders have been reported.3-12 Alterations in platelet membranes have been studied to relate these

with possible abnormalities in the brain.13-16 However, the reports in the literature are conflicting, contradictory and equivocal.4,5,8-12,16-18 No clear-cut pointer or a diagnostic biochemical parameter has been identified thus far. The incidence of affective disorders is very high. It has been reported that in the United States about 15% of the population suffers from mood disorders.1 This results in considerable loss of productivity. The World Health Organization (WHO) in its 1997 annual health report indicated that most persons with severe activity limitation suffered from mood disorders; the estimated number was 146 million.19 In the light of the above, it is highly desirable to search for a parameter which can be useful for the diagnosis, prognosis and for monitoring the recovery of mood disordered patients undergoing treatment. It is also desirable that the procedure should be minimally invasive. With these objectives in mind we decided to quantitate characteristics of the erythrocyte membranes from unipolar and bipolar patients in comparison with age and sex matched controls.

Correspondence: Prof. S.S.Katyare, Department of Biochemistry, Faculty of Science, M.S. University of Baroda, Vadodara, India, 390 002 e-mail: [email protected]

METHODS Patient selection Diagnoses were made by the committee comprising the head, psychiatry department, other doctors attached to the ward and RMOs 11

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in the panel, of the Civil Hospital, Ahmedabad, Gujarat, India. The patients were diagnosed through clinical interview (DSM IV criteria). Collateral information was obtained from their family members. All the subjects were married, none were single, divorced or widow(er) and they all belonged to the middle income group. None of them were suffering from any other diseases. Sample collection Blood samples were collected from mood disorder patients and agematched control volunteers with informed consent in heparinized vials. The unipolar and bipolar subjects were either from the Out Patients Department of Psychiatry Ward, Civil Hospital, Ahmedabad, Gujarat, India, or those who were admitted in the ward for treatment. Control subjects were randomly selected volunteers from the Department of Biochemistry, Faculty of Science, M.S.University of Baroda, Vadodara, Gujarat, India. Isolation of erythrocyte membranes Erythrocyte membranes were isolated essentially according to the method of Hanahan and Ekholm.20.21 Briefly, the blood samples were centrifuged at 475 X g for 8 min to separate the cells and the plasma. The plasma was carefully decanted and was used as the source for the measurement of BChE activity. The buffy coat overlaying the RBC pellet was discarded and the pellet was washed twice with 0.9 % NaCl. The washed RBCs were then subjected to hypotonic lysis in 14 mM Tris HCl buffer pH 7.4 at 0-4°C. The lysate was centrifuged at 30,000 X g for 35 min and the supernatant was discarded. The pellet was washed repeatedly with the same buffer to obtain hemoglobin-free membranes. The final pellet was resuspended in the same buffer at a concentration of about 1mg protein/ ml. Lipid analysis Extraction of total lipids, separation of phospholipid classes by thin layer chromatography and estimations of cholesterol and total phospholipid phosphorous were according to the methods cited.22-26 Membrane fluidity The fluidity of erythrocyte membranes was determined at 25 °C using DPH as the probe, in a RF 5000 Shimadzu spectrofluoriphotometer.27 Enzyme assays ATPase The Na+, K+ ATPase activity was measured in the medium (total volume: 0.4 ml) containing 50 mM Tris HCl buffer pH 7.4, 100 mM NaCl, 10 mM KCl and 4 mM MgCl2. 50-100 ìg of erythrocyte membrane protein was used as the source of the enzyme. After pre-incubation at 37°C for 5 min the reaction was initiated by adding 1.25 mM ATP. The reaction was carried out for 90-120 min and terminated by adding 0.1 ml 5 % (w/v) of SDS.22 Estimation of liberated inorganic phosphorus was according to the method of Fiske and Subba Row.28

AChE and BChE activity The activities were measured essentially according to the procedure of Ellman et al. as described previously.22,29 Thus for the measurement of AChE activity, the assay system (total volume: 1 ml) contained 100 mM potassium phosphate buffer pH 8.0, 0.32 mM DTNB, 0.1 mM ETPZ.HCl and 10-50 mg of the erythrocyte membrane protein as the source of the enzyme. After

pre-incubation at 37 °C for 1-2 min the reaction was initiated by adding the substrate ACTI and the linear rate of increase in absorbance at 412 nm was recorded over a period of 60 to 90 seconds. For determination of BChE activity the assay system was essentially the same as that for AChE except that the buffer was 50 mM Tris HCl pH 8.0 and ETPZ.HCl was omitted; BCTI was the substrate. 20 ml of 1:10 diluted plasma was used as the source of the enzyme. For substrate kinetics analysis the concentration of substrates (ACTI/ BCTI in the two assay systems respectively) was varied from 25 ìM to 10 mM. For temperature kinetics studies the enzyme activity was determined over a temperature range of 5 to 53 °C with an increment of 4 °C at each step. Measurements were carried out at substrate (ACTI/ BCTI) concentration of 5 mM. The data for substrate kinetics were analyzed by the LineweaverBurk, Eadie-Hofstee and Eisenthal and Cornish-Bowden methods for determination of Km and Vmax.30 The values of Km and Vmax obtained by the three methods were in close agreement and were averaged. The results are given as mean ± SEM of the averaged values. The ranges for individual mean value are indicated in the parentheses. The data on temperature kinetics were analyzed for determination of energies of activation in the high and low temperature ranges ( E1 and E2 respectively) and phase transition temperature (Tt) .31 All the kinetics data were computer analyzed employing Sigma plot version 5.0.21,22,30 Protein estimation was according to the method of Lowry et al. with bovine serum albumin used as the standard.32 Chemicals ACTI, BCTI, ETPZ.HCl and DPH were purchased from Sigma Chemical Co. U.S.A. Sodium salt of vanadium free ATP and DTNB were purchased from SRL, India. Silica Gel G was from E. Merck, Germany. All other chemicals were of analytical re-agent grade and were purchased locally. Statistics Statistical evaluation of the data was by Student’s t-test. Comparisons among the groups were by analysis of variance (ANOVA). RESULTS The details regarding the age, sex, duration of mood disorder and treatment for the 9 unipolar patients and 12 bipolar I patients are given in Table I. Of the patients listed in the unipolar group # 5 was certified by the psychiatrists as “cured”, since this patient had been asymptomatic for the last one and half year. The patient suffered from headache and the family members advised him to visit the psychiatry ward (where he had been admitted two years ago) rather than going to a general practitioner. [We met this patient by chance and he agreed to participate in the study.] The patients were either admitted in the hospital or were visiting the OPD for further treatment. Thus most of these patients (except patients # 1 and # 2 in bipolar group and #1 in unipolar group) were on drug therapy. The unipolar patients received treatment with tricyclic anti-depressants e.g. amitriptyline or benzodiazepam e.g. lorazepam. The patients in bipolar group received lithium treatment. Patient # 11 received megitol and largacril. Mean age of the patients in the unipolar group was 34.8± 3.5 years whereas that of patients in the bipolar group was 33.1±3.4 years. The mean age of the control subjects was 32.0± 3.0 years. 14

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Table II. Erythrocyte membrane TPL, CHL and TPL/CHL (mole: mole) ratio in mood disorder patients.

Table I. Case histories of unipolar/ bipolar patients. Group

Unipolar

Bipolar

Patient number

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12

Age (Y)

35 32 27 32 57 21 34 28 52 30 22 25 34 40 55 52 39 20 36 25 21 28

Sex

Duration TPL

F F M F M F F M M F F M F M F F M F M M F M

First Symptom observed/diagnosed

Drug taken

20 Days 1 Months 5 Years 17Months 2 Years 6 Years 3 Years 1 Year 6 Years 3 ˚ Years — 2 Months 4 Months 6 Months 8 Months 1 Year 3 Years 3 Years 4 Years 5 Years 6 Years 13Years

— 4 Days 5 Years 17 Months 2 Years 6 Years 3 Years 1 Year 6 Years 3 ˚ Years — — 4 Months 6 Months 8 Months 1 Year 3 Years 3 Years 4 Years 5 Years 6 Years 13 Years

The mean duration from first symptoms to study entry was 2.8± 0.7 years and 3.3±1.1 years for unipolar and bipolar I groups respectively with the mean duration of treatment 3.1±0.7 and 3.6±1.2 years respectively for the two groups. The unipolar group included 4 males and 6 females while the bipolar group consisted of 6 males and 6 females. Control groups comprised of 11 males and 5 females. In view of the large variations in the duration of the mood disorder and of drug treatment (e.g. see Table I) the final results of analysis are given for a group as a whole and the values are represented as mean ± SEM. The data in Table II show TPL and CHL content of erythrocyte membranes and the molar ratios of TPL/CHL. From the data in Table II it can be noted that the TPL content increased significantly in the unipolar patients. A similar trend was seen even in the bipolar group. However, the increase was of a lesser magnitude and the increase was not statistically significant. The CHL content was significantly high ( 2.3 and 2 folds higher respectively) in the unipolar and bipolar patients. Once again the magnitude of the increase was much higher in the unipolar group. Consequently TPL/CHL (mole: mole) ratio was significantly low, more so in the unipolar group. {It may be mentioned here that the data for patient # 5 in unipolar group who was certified as “ cured” are not included in Table II and also that in the patient #5 TPL/CHL ratio was normalized to 1.0} Thus disproportionate increase in membrane TPL and CHL, and significantly decreased TPL/CHL ratio seem to be the characteristic feature of unipolar patients. For the bipolar patients increase only in CHL content and decreased TPL/CHL (mole: mole) ratio may be a distinguishing feature. Analysis of variance revealed that the differences in the groups with respect to the three parameters, TPL, CHL and their molar ratios were highly significant. It has been reported that the erythrocytes have a characteristic methylase, which is responsible for conversion of PE to PC.33,34 It has been further reported that the methylase activity decreased in mood disorders thereby resulting in decreased PC content. In the

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TPL/CHL (mole: mole)

Control (12)

516.17±28.50 (376.55-723.71)

238.72±12.83 (191.1-303.11)

1.082±0.039 (0.927-1.314)

Unipolar (8)

832.00±58.99*** (563.51-1062.01)

545.96±52.91*** (396.00-766.56)

0.775±0.039*** (0.688-0.794)

Bipolar (10)

691.44±149.60 (301.34-988.98)

480.03±112.53* (167.12-460.53)

0.866±0.043** (0.645-0.992)

p< 0.01

p< 0.01

p< 0.01

Significance by ANOVA

The experimental details are as given in the text. The results are given as mean ± SEM of the number of observations indicated in the parentheses. * p < 0.05, ** p < 0.002 and *** p < 0.001 compared to control.

light of this observation it was of interest to find out if the membrane phospholipid composition had altered in these affective disorders. These data are shown in Table III. It is clear that there was no change composition-wise in any of the phospholipid class except for PA, which increased in both unipolar and bipolar patients. Comparison of groups by ANOVA showed that the difference was highly significant. It is possible that the increased PA contents in the two mood disorder groups may relate to altered phospholipid turnover. Since CHL is one of the determinants of the membrane fluidity/ rigidity35 it was of interest to find out if the membrane fluidity is altered in mood disorders. The data in Table IV show that indeed the membranes were somewhat more rigid in the two affective disorders although the values in the unipolar group did not reach a significance level by Student’s t-test. However, the group analysis showed that the difference amongst the groups was significant at p< 0.05. We next examined the erythrocyte membrane Na+, K+ ATPase and AChE, and plasma BChE activities. The results are given in Table V. As can be noted, the Na+, K+ ATPase activity was unchanged. Even analysis by ANOVA showed that the difference among the groups was not significant. Therefore Na+, K± -ATPase may not be a good marker. Our results are thus consistent with the observation of earlier researchers.4,17 The AChE activity was high in both unipolar as well as bipolar groups. However, the increase was statistically Table III. Phospholipid composition of erythrocyte membranes in mood disorder patients. Phospholipid composition ( % of total)

Lyso SPM PC PI PS PE PA

Control (10)

Unipolar (9)

Bipolar (11)

2.87 ± 0.78 23.44 ± 1.06 30.99 ± 1.19 5.05 ± 0.77 6.93 ± 0.84‘ 25.28 ± 1.11 5.32 ± 0.83

3.45 ± 0.57 22.15 ± 1.55 30.10 ± 1.01 5.13 ± 0.39 5.07 ± 0.69 25.15 ± 2.14 8.22 ± 1.28

3.25 ± 0.81 23.88 ± 1.19 29.85 ± 1.01 3.76 ± 0.76 4.93 ± 0.92 24.55 ± 1.07 a 9.50 ± 1.3*

The experimental details are as given in the text. The results are given as mean ± SEM of the number of observations indicated in the parentheses. * p < 0.02 compared to control. a Significance by ANOVA p< 0.01

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Table IV. Fluidity parameters of erythrocyte membranes in mood disorders. Fluidity parameter Fluorescence polarization, p

Fluorescence anisotropy, r

Limited hindered anisotropy, ra

Order parameter, s

Control (16)

0.350 ± 0.005 (0.304-0.382)

0.264 ± 0.004 (0.226-0.296)

0.253±0.006 (0.201-0.295)

0.799±0.009 (0.713-0.864)

Unipolar (9)

0.377±0.022 (0.296-0.478)

0.288±0.02 (0.219-0.379)

0.285±0.026 (0.192-0.405)

0.842±0.039 (0.697-1.013)

Bipolar (12)

0.375±0.006* (0.332-0.399)

0.286±0.007** (0.249-0.307)

0.282±0.007* (0.232-0.309)

0.844±0.010*** (0.766-0.885)

p< 0.05

p< 0.05

p< 0.05

p< 0.05

Significance by ANOVA

The experimental details are as given in the text. The results are given as mean ± SEM of the number of observations indicated in the parentheses. * p < 0.01, ** p < 0.002 and *** p < 0.001 compared to control. +

+

Table V. Erythrocyte membrane Na , K ATPase and AChE and plasma BChE activities in mood disorder patients. +

+

Na , K ATPase (mmole Pi/hr/mg protein)

AChE (mmole ACTI hydrolyzed/ min /mg protein)

BChE (mmole BCTI hydrolyzed /min /ml plasma)

Control

211.00±28.34 (122.29-376.80)

0.662±0.082 (0.223-0.996)

4.98±0.22 (3.80-6.67)

Unipolar

247.0±50.24 (80.09-425.08)

1.08±0.062** (0.840-1.343)

6.28±0.40* (4.87-8.28)

Bipolar

287.3±50.00 (108.86-494.9) N.S

0.90±0.100 (0.588-1.273) p< 0.01

6.24±0.60 (4.21-7.11) p< 0.01

Significance by ANOVA

The experimental details are as given in the text. The results are given as mean ± SEM of 8-12 independent observations. * p < 0.01 and ** p< 0.002 compared to control.

significant only for the unipolar group. The differences in the groups were highly significant when evaluated by ANOVA. Likewise the plasma BChE activity also increased significantly in the unipolar group. A similar increase was noted even for the bipolar group, although the difference was not statistically significant compared to the controls. Comparison of groups by ANOVA showed that the difference was highly significant. We have previously reported that the human erythrocyte membrane AChE has two kinetically distinguishable components while plasma BChE has three components.21 It was therefore of interest to find out if the observed changes i.e. increase in the erythrocyte membrane AChE and the plasma BChE in both the mood disorders could be traced to the individual components. To elucidate this possibility we examined the substrate saturation kinetics. The typical substrate saturation curves for the AChE are shown in Fig. 1. The corresponding Eadie-Hofstee plots are also included. The values for the Km for the two components (Km1 and Km2) and the corresponding Vmax (V max1 and V max2) values are given in Table VI. As can be noted, in the unipolar patients the value of Km2 increased almost by 2.5 folds whereas in the bipolar group the V max1 had decreased by 40 %. As evaluated by ANOVA, difference among the groups were highly significant. The observed changes may relate to altered membrane compositions (e.g. see Tables II and III). AChE is known to be a membrane-bound enzyme and it has

been reported that its kinetic properties change after binding to membrane.36 In view of the altered TPL and CHL contents in the erythrocyte membranes (Table II) it was of interest to find out whether these alterations influenced the temperature kinetics of AChE. The typical plots depicting the temperature-dependentchanges in AChE activity are shown in Fig. 2. The corresponding Arrhenius plots are also included. It is self evident that the patterns for the two mood disorder groups differ considerably from that of the control. Thus one notes that in the case of the control group AChE activity shows a sharp rise after around 40°C as compared to a progressive steady increase in the two mood disorder groups; the changes are also reflected in the corresponding Arrhenius plots. Thus in the control groups the Arrhenius plot is biphasic and the values of E1 and E2 were 72.6 and 37.4 KJ/mole and the phase transition temperature was around 40°C. This agrees well with our previously reported values.21 By contrast, in the two affective disorders the Arrhenius plots were monophasic and the phase transition was abolished. The energies of activation were 37.4 and 45.6 KJ/mole respectively in the two groups (Table VII). The values of energies of activation were comparable to E2 in the control groups, and this value in the bipolar group was significantly high. Comparison of groups by ANOVA reveled that the differences were significant. Obviously the changes correlate with and reflect the membrane lipid alterations. However, in case of the bipolar patients # 3 and # 4 who were suffering from this disorder for 4-6 months the Arrhenius plots were biphasic (data not shown). This would suggest that the membrane

Fig.1. Typical substrate saturation curves for human erythrocyte membrane AChE in (a) control, (b) unipolar and (c) bipolar patients. The respective Eadie-Hofstee plots are shown in (d), (e) and (f). Experimental details are as given in the text. For determination of substrate kinetics of AChE, ACTI was used as a substrate over a concentration range of 0.025 to 10mM. The abscissa represents the reaction velocity v, while the ordinate represents [S] and v/[S] ratio for substrate saturation curves and the Eadie-Hofstee plots respectively. Reaction velocity v -1 -1 = mmol of ACTI hydrolyzed min mg protein . V/[S]= reaction velocity divided by the corresponding substrate concentration.

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alterations occur at later stages and are sustained despite drug treatment for long duration. Component I Component II Since the pattern for the temperature-dependentKm1 Vmax 1 Km 2 Vmax 2 changes in AChE activity differed significantly in the two mood disorders (Fig.2), we decided to comControl(10) 0.073±0.008 0.693±0.069 0.490±0.074 1.275±0.125 pare the activities of the AChE at 25°C, 37°C and ( 0.041-0.126 ) ( 0.406-1.015 ) ( 0.206-0.826 ) ( 0.772-1.847 ) 53°C the latter being the highest temperature we have Unipolar (8) 0.089±0.017 0.685±0.052 1.212±0.198** 1.150±0.043 employed in our studies. These data are shown in ( 0.038-0.163 ) ( 0.490-0.914 ) ( 0.450-1.550) ( 0.940-1.270 ) Table VIII. We then decided to evaluate the data by taking the activity coefficient ratios of two temperaBipolar (10) 0.073±0.009 0.412±0.057* 0.415±0.078 1.029±0.129 ture groups i.e. 53°C/25°C and 53°C/37°C. These ( 0.026-0.125 ) ( 0.141-0.699 ) ( 0.319-0.516 ) ( 0.471-1.690) Significance by ANOVA N.S p< 0.01 p< 0.01 N.S values are also included in Table VIII. As can be noted, the values of the activity coefficients were The experimental details are as given in the text. The results are given as mean ± SEM of the number of obser6.88 and 3.92 respectively in the control group. Invations indicated in the parentheses. Substrate kinetics measurements of RBC membrane AChE were carried out terestingly both the values decreased significantly using ACTI as the substrate over the concentration range of 0.025 to 10 mM. in the unipolar group whereas activity coefficient Units: Km= mM, Vmax = m mol of ACTI hydrolyzed min-1 mg protein-1. * p < 0.02, ** p < 0.01 compared to the corresponding controls. corresponding to the latter value only had decreased in the bipolar patients. The observed differences were also found to be highly significant also by ANOVA. Our results therefore suggest that determination of AChE activity at three given temperatures and the activity coefficient ratios could serve as good diagnostic pointers. The typical substrate saturation curves and the corresponding Eadie-Hofstee plots for plasma BChE are shown in Fig. 3. As is evident, consistent with our earlier observation three kinetically different and distinguishable components of BChE are noted in the plasma of control group. A similar pattern is seen also for the two mood disorder groups. The Km values for the three components were comparable in all the groups. The Vmax values tended to be higher in the mood disorder groups. However, these latter differences were not statistically significant. ANOVA revealed that the groups differed only with respect to V max2 (Table IX). We then determined the temperature dependence of BChE activity. The Typical plots are shown in Fig. 4. The corresponding Arrhenius plots are also included. It is evident that in the mood disorder patients, at any given temperature, the BChE activity was higher than that in the control group. The Arrhenius plots were monophasic Fig. 2. Typical temperature curves for AChE from human erythrocyte in (a) constraight lines and the energies of activation were 22.58±0.87, trol, (b) unipolar and (c) bipolar patients. The respective Arrhennius plots are 23.21±1.36 and 25.22±0.73 KJ/mole respectively for control and shown in (d), (e) and (f). Experimental details are as given in the text. The AChE the two mood disorder groups. A slight variation in the energies of activity in RBC membranes was determined with 5 mM ACTI. The abscissa represents the log of reaction velocity v, while the ordinate represents reciprocal activation as observed in the two mood disorder groups may be atof absolute temperature T*1000. Reaction velocity v = m mol of ACTI hydrolyzed tributed to the possible compositional changes (discussed below). -1 -1 Table VI. Substrate kinetics properties of erythrocyte membrane AChE in mood disorders.

min mg protein . Absolute temperature T= °Kelvin.

DISCUSSION The present studies were undertaken to examine erythrocyte membrane functions in mood disorders in compariTable VII. Arrhennius kinetics analysis of erythrocyte membrane AChE in mood disorders. son with those from normal volunteers. The objective of these studies was to look for a suitable biochemical diag(Energy of activation, KJ/mole) Phase transition temperature, E1 E2 Tt (°C) nostic marker. Indeed our studies have brought forth the subtle differences in erythrocyte membrane function in the Control (12) 72.55 ± 5.43 37.36 ± 3.36 39.9 ± 1.09 two mood disorder groups. Thus, the TPL content increased (59.63-101.30) (20.55-44.39) (36.25-43.59) only in the unipolar group while CHL content increased Unipolar (8) — 37.40 ± 3.44 — in both the groups. Consequently the TPL/CHL molar ratio (22.18-53.97) was always low in the two mood disorder groups compared with the controls. Most interestingly, in patient # 5 Bipolar (10) — 45.64 ± 3.83* — in unipolar group (who was certified as cured) the TPL/ (25.99-69.70) CHL molar ratio returned to normal value. Significance by ANOVA p< 0.05 Earlier, it has been reported that in mood disorder the The results are given as mean ± SEM of the number of observations indicated in the parentheses. PC content decreased.3 However, we could not detect any Enzyme activities were determined with 5 mM ACTI concentration. change in the PC component. This may possibly be attrib* p < 0.05 compared with control. uted to the fact that the drug treatment for 2 weeks is re17

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Table VIII.Erythrocyte membrane AChE activities at 25°C, 37°C and 53°C in mood disorder patients. Activity (m mol of ACTI hydrolyzed -1 -1 min mg protein ) 25°C 37 °C

53°C

Activity coefficient ratio (53°C/25°C) [53°C/37°C]

Control (11)

0.267±0.042

0.572±0.092

1.849±0.301

6.877±0.452

3.915±0.178

Unipolar (8)

0.525±0.045****

0.823±0.067

1.908±0.176*

3.785±0.363****

2.365±0.172****

Bipolar (12) Significance by ANOVA

0.430±0.047** p< 0.01

0.869±0.115 p< 0.01

2.621±0.419 p< 0.01

6.463±0.685 p< 0.01

2.909±0.197*** p< 0.01

The results are given as mean ± SEM of the number of observations indicated in the parentheses. Enzyme activities were determined with 5 mM ACTI concentration. * p < 0.05, ** p

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