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Increased phospholipase A2 activity in schizophrenia with absent response to niacin
Schizophrenia Research 61 (2003) 1 – 6 www.elsevier.com/locate/schres
Increased phospholipase A2 activity in schizophrenia with absent response to niacin Hildeberto Tavares Jr., Juliana Yacubian, Leda L. Talib, Na´dia R. Barbosa, Wagner F. Gattaz* Laboratory of Neuroscience, Department and Institute of Psychiatry, Faculty of Medicine, University of Sa˜o Paulo, Rua Dr. Ovı´dio Pires de Campos s/n, P.O. Box 3671, 05403-010 Sa˜o Paulo-SP, Brazil Received 16 November 2001; received in revised form 8 February 2002; accepted 14 February 2002
Abstract An absent response to the niacin skin test has been reported to occur in about 80% of schizophrenic patients, as compared to 20% of healthy individuals. Niacin provokes redness in skin caused by a capillary vasodilatation mediated by prostaglandins. The metabolism of prostaglandins is regulated by the enzyme phospholipase A2 (PLA2). Several studies have reported increased PLA2 activity in schizophrenia. In this study we investigated the relationship between niacin response and PLA2 activity in 38 drug-free schizophrenic patients and in 28 healthy controls. Twenty-two of these patients were reevaluated after 8 weeks under treatment with new generation antipsychotic drugs. Niacin response was absent in 23% of the schizophrenic patients and in 14% in controls (n.s.). PLA2 activity was higher in schizophrenics than in controls (344 F 115 vs. 290 F 71 pmol/ml/min; p = 0.03). Patients with absent response to niacin had the highest PLA2 activity as compared to those with positive response (426 F 155 vs. 319 F 111; p = 0.02). After 8 weeks on antipsychotic treatment, PLA2 activity was reduced (355 F 115 before, 267 F 39 after, p = 0.001) and 4 out of 13 patients with absent response to niacin converted to positive. The reduction of PLA2 activity in these patients was higher than in patients who remained with absent response (36% vs. 23%). Our data support the findings that absent response to niacin is more frequent in schizophrenic than in healthy individuals although the magnitude of the difference was smaller than that reported in the literature. The relationship between absent response to niacin in schizophrenia and increased PLA2 activity suggests further that the skin test may be useful to easily identify a subgroup of patients with a disordered phospholipid metabolism. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Niacin test; Phospholipase A2 activity; Schizophrenia; Antipsychotic drugs
1. Introduction Niacin is a water-soluble vitamin B that causes skin redness when administrated orally or topically to individuals (Murrell and Taylor, 1959). This response is secondary to the liberation of prostaglandin D2 by skin *
Corresponding author. E-mail address: [email protected] (W.F. Gattaz).
macrophages (Morrow et al., 1992). Decreased or absent response to oral niacin has been reported in schizophrenic patients (Horrobin, 1980; Fiedler et al., 1986; Rybakowski and Weterle, 1991). Ward et al. (1998) found 83% of nonresponse to cutaneous administration of niacin (aqueous methyl nicotinate) vs. 23% in healthy controls. Such results have been confirmed by other groups (Easton et al., 2000; Shah et al., 2000), suggesting that niacin response could indicate a bio-
0920-9964/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0920-9964(02)00281-5
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H. Tavares Jr. et al. / Schizophrenia Research 61 (2003) 1–6
logically more homogeneous subgroup of schizophrenic patients. Phospholipase A2 (PLA2) is an enzyme that cleaves fatty acids from the sn2-position of phospholipids in cellular membranes, liberating free fatty acids and lisophospholipids. Arachidonic acid (AA) is produced in this process and generates prostaglandins by the action of cyclo-oxygenase. Increased PLA2 activity has been found in serum, plasma, platelets and brain tissue from schizophrenic patients (Gattaz et al., 1987, 1990, 1995; Ross et al., 1997), although negative results were also reported by one study (Albers et al., 1993). In face of the modulation of prostaglandin synthesis by PLA2, we investigated, in the present study, the relationship between the enzyme activity and niacin response in schizophrenia.
2. Material and methods 2.1. Subjects and clinical assessment The study was performed at the Department and Institute of Psychiatry of the University of Sa˜o Paulo. The sample comprises of 38 schizophrenic patients (age 32.5 F 10.3 years; 19 male, 19 female) and 28 healthy individuals (age 30.5 F 8.2 years; 11 male, 17 female). All individuals were included in the study after having signed an informed consent. To be included in the study, subjects should be between 18 and 60 years old and should not have taken any analgesic or antiinflammatory drugs the last 2 days. Individuals with diabetes, rheumatological or dermatological diseases were not included in the study. Controls were not under any psychotropic medication and could not have any first degree relative with schizophrenia. Schizophrenic patients were diagnosed using the Structural Clinical Interview for DSM-IV (American Psychiatric Association, 1994) and should score at least 60 in the Positive and Negative Symptoms Scale for Schizophrenia— PANSS (Kay et al., 1987). All patients (n = 38) were from the community and had been ill for 7.8 F 6.8 years and had been previously hospitalized 2.3 F 1.3 times; all of them were drug-free for at least 2 weeks (mean 43.6 F 62.6 weeks) and 19 were drug naı¨ve. The mean total PANSS score for the sample was 82.4 F 20.0 and the sub-scores were 20.1 F 4.8 for positive symptoms; 27.2 F 9.9 for neg-
ative symptoms and 34.7 F 10.9 for general psychopathology. Twenty-two of the schizophrenic patients were reevaluated after 8 weeks of treatment with new generation antipsychotics (14 with olanzapine mean daily doses of 9.5 F 7.5 mg, 6 with amisulpride 500 F 300 mg and 2 with risperidone 6 mg). 2.2. Niacin test Niacin skin test was performed applying solutions of aqueous methyl nicotinate (0.1, 0.01, 0.001 and 0.0001 M) to four corresponding pieces of filter paper (2 2 cm) attached to the forearm skin with a nonallergic adhesive plastic strip. The strip was let in contact with the skin for 5 min and the response was evaluated every 5 min from 5 to 30 min, without washing niacin solution from the skin. The flush response was then scored as 0 = no erythema, 1 = incomplete erythema, 2 = complete erythema or 3 = erythema plus edema (Ward et al., 1998). Scores 0 and 1 were considered absent response and scores 2 and 3, positive. 2.3. PLA2 determination Serum was obtained from 10-ml blood collected in glass tubes without additions, kept at room temperature. It was immediately centrifuged in 3000 rpm for 15 min. Aliquots of 30 Al were frozen ( 70 jC). PLA2 activity in serum was then determined by a fluorimetric assay based on the procedure used by Thuren et al. (1985), with the modifications described bellow: 2.3.1. Chemicals 1-Octacosanyl-2-(1-pyrenehexanoyl)-sn-glycero-3phosphomethanol ammonium salt (C28-O-PHPM) and (pyren-1-yl) hexanoic acid (Molecular Probes, USA); n-heptan, toluene; ethanol; methanol; chloroform; hydrogen chloride and potassium carbonate (Merck, Germany); Trizma type pH 7.4 (Sigma USA); sodium borate (Reagen, Brazil); all chemicals were of analytical grade. 2.3.2. Preparation of substrate Dilute the C28-O-PHPM in toluene/ethanol (1:2, v/v) to obtain a concentration of 280 nmol/ml (stock solution). Evaporate 225 nmol/ml of this solution under a gentle stream of nitrogen and dissolve the residue in 800
H. Tavares Jr. et al. / Schizophrenia Research 61 (2003) 1–6
Al of ethanol (ethanolic solution). Rapidly add this solution into a test tube containing 5.6 ml of Tris (hydroxymethyl) aminomethane (Tris) – hydrogen chloride buffer (20 mmol/l, pH 7.4) (working solution). Keep the C28-O-PHPM solution on ice. 2.3.3. Assay procedure Add into a glass test tube 160-Al working solution of substrate and 40-Al serum, using as blank Tris – hydrogen chloride buffer (20 mmol/l, pH 7.4). Vortex both thoroughly and incubate at 37 jC in the dark in a shaking water bath for 60 min. Stop the reaction by adding 1.3 ml chloroform/methanol: n-heptan, (1.25:1.41:1, v/v/v) and vortex thoroughly for 30 s. Add 300-Al sodium borate– potassium carbonate buffer (0.14 mol/l each, pH 10.5) and thoroughly mix the solution. Centrifuge the tubes for 10 min at 2000 g at room temperature. Pipette 750 Al of the aqueous phase into a magnetically stirred four-window quartz cuvette and add 750-Al 40% methanol solution. Set fluorescence excitation at 343 nm and measure emission at 400 nm. 2.3.4. Instrument calibration Adjust the fluorescence intensity to 1 106 readings/s when the cuvette contains 50 pmol of (pyren-1yl) hexanoic acid in 40% methanol solution and 750 Al of the aqueous phase of control serum (volunteer)— final volume: 1.5 ml. We measured fluorescence with a fluorescence spectrometer with two emission subsystems (Photon Technology International—Canada) and for the entrance and exit sides of both the excitation and emission monochromators (2.5 turns of the slit micrometers—bandpass 5 nm) and excitation wavelength: 343 nm and emission wavelength: 400 nm, for both channels. PLA2 activity is expressed as picomoles of free (pyren-1-yl) hexanoic acid produced per minute per milliliter of serum and is calculated as follows: PLA2 activity ðpmol min1 ml1 Þ ¼
I 25 1:33 1:54 0:00005 60 0:040
where: I = fluorescence intensity, in arbitrary units read from spectrofluorometer; 25 = correction factor to obtain the activity per milliliter of serum; 1.33 = adjust-
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ment to whole aqueous phase; 1.54 = recovery of the substrate is estimated as 65%; 60 = incubation time (min); 0.040 = volume of serum (ml); 0.00005 = conversion of the fluorescence intensity reading into picomoles of (pyren-1-yl) hexanoic acid. Statistical analysis was performed with chi-square test for the analyses of niacin categorical data and two-tailed t test for PLA2 activity.
3. Results Absent response to topic niacin in schizophrenia was more frequent than in controls for most of the concentrations and measurement times (Table 1). However, a trend for significance was found only for the concentration of 0.001 M at 10, 25 and 30 min. To allow comparisons with other reports that used the same methodology as in the present study, we will analyze the data related to the niacin concentration of 0.01 M at 10 min (Ward et al., 1998). Although the control individuals and the schizophrenic patients were not matched by gender, no significant statistical differences were found in niacin test comparing the groups separated by sex. We also did not find differences in niacin test between the previously medicated (n = 19) and the drug naı¨ve patients (n = 19). No significant differences in psychopathological scores and clinical improvement were found between
Table 1 Absent response to niacin (%) in schizophrenic patients and in healthy controls
0.01 M 5 min 0.01 M 10 min 0.01 M 15 min 0.01 M 20 min 0.01 M 25 min 0.01 M 30 min 0.001 M 5 min 0.001 M 10 min 0.001 M 15 min 0.001 M 20 min 0.001 M 25 min 0.001 M 30 min
Schizophrenia (n = 38) (%)
Control (n = 28) (%)
p
Chi
60 23.7 10.5 10.5 18.4 18.4 100 86.8 84.2 84.2 84.2 86.8
57.1 14.2 14.2 14.2 17.2 17.2 96.4 67.8 67.8 67.8 64.2 64.2
0.49 0.26 0.46 0.46 0.54 0.54 0.62 0.06 0.10 0.10 0.06 0.03
0.08 0.9 0.21 0.21 0.05 0.05 0.05 3.48 2.46 2.46 3.48 4.67
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Table 2 PANSS scores in 22 schizophrenic patients according to the response (positive or absent) after 10 min of niacin 0.01 M
Total PANSS Positive PANSS Negative PANSS
Absent niacin (n = 9)
Positive niacin (n = 13)
p
t
90.8 F 28.2 19.0 F 4.8 30.8 F 8.7
80.0 F 17.3 20.4 F 4.9 26.1 F 10.2
0.1 0.86 0.71
0.82 0.57 1.01
Table 4 PLA2 activity in 38 schizophrenic patients compared to 28 controls; in 22 schizophrenic patients before and after 8 weeks of treatment with new generation antipsychotics; and in 9 schizophrenic patients with absent response to niacin compared to 13 patients with positive response to niacin (after 10 min of niacin 0.01 M) Schizophrenia (n = 38)
Control (n = 28)
p
t
344 F 115
290 F 71
0.03
2.18
Before Treatment (n = 22) After Treatment (n = 22)
patients with absent and positive response to niacin, using t test for comparison of means (Table 2), in any niacin concentration or time of evaluation. After 8 weeks on antipsychotic drug treatment, 4 of the 13 patients who were at baseline were niacin-negative turned into positive ( p = 0.06, Table 3). PLA2 activity (Table 4) was increased in schizophrenics as compared to controls (344 F 115 vs. 290 F 71 pmol/ml/min; p = 0.03; t = 2.18). PLA2 activity decreased by 25% in the 22 patients reevaluated after 8 weeks on antipsychotic treatment (355 F 11 before and 267 F 39 after, p = 0.001; t = 4.04). No correlations were found between PLA2 activity at baseline nor PLA2 reduction after treatment with psychopathological scores and improvement. There was not any correlation between PLA2 activity and the drug-free period (Spearman = 0.10, p = 0.57). Patients with absent response to niacin (n = 13) had higher PLA2 activity than those with positive (n = 9) response (426 F 155 vs. 319 F 111, p = 0.02; t = 2.3).
Table 3 Absent response to niacin (%) at different times and concentrations in 22 schizophrenic patients before and after 8 weeks on antipsychotic drug treatment
0.01 M 5 min 0.01 M 10 min 0.01 M 15 min 0.01 M 20 min 0.01 M 25 min 0.01 M 30 min 0.001 M 5 min 0.001 M 10 min 0.001 M 15 min 0.001 M 20 min 0.001 M 25 min 0.001 M 30 min
Before treatment (%)
After treatment (%)
p
Chi
60.0 22.7 4.5 9.0 13.6 13.6 86.3 86.3 81.8 81.8 77.3 81.8
40.9 4.5 4.5 9.0 13.6 13.6 77.2 77.2 68.2 68.2 68.2 68.2
0.03 0.06 0.90 0.17 0.04 0.03 0.12 0.12 0.07 0.07 0.16 0.07
5.5 5.8 4.2 4.4 8.3 8.2 3.8 3.8 4.2 4.2 2.37 4.2
355 F 11
267 F 39
Absent Niacin (n = 9)
Positive Niacin (n = 13)
426 F 155
319 F 111
0.001 4.04
0.02
2.3
The patients who converted niacin response (0.01 M after 5 min) from absent into positive after treatment (n = 4) had a 36% reduction in PLA2 activity, against 23% in those who remained nonresponsive (n = 5).
4. Discussion Absent flushing in response to niacin was more frequent in schizophrenic patients than in controls in our sample, but the magnitude of the difference was less than that reported by other groups. Using a similar niacin test as in our study, for the concentration of 0.01 M, Ward et al. (1998) found 83% absent response in drug-treated schizophrenic patients and 23% in controls. Using a quantitative analysis, Shah et al. (2001) found less skin flushing in schizophrenic patients. We are not able to explain these differences among the results, as similar test procedures were used in those studies. Puri et al. (2000) found a predominance of negative symptoms in schizophrenic patients with absent response to niacin. In our sample, we could not replicate this relationship between niacin response and psychopathology scores (Table 2), which was also not observed in other studies (Glen et al., 1996; Ward and Glen, 2001). In line with previous studies (Gattaz et al., 1987; Ross et al., 1997), PLA2 activity in serum was increased in drug-free schizophrenia and the enzyme activity was reduced after antipsychotic drug treatment. It has already been shown that haloperidol (Gattaz et al., 1987) and other first generation anti-
H. Tavares Jr. et al. / Schizophrenia Research 61 (2003) 1–6
psychotics (Trzeciak et al., 1995) reduce PLA2 activity. The present results extend those findings also to new generation antipsychotics such as olanzapine, amisulpride and risperidone. Reduced PLA2 activity trough the action of antipsychotic drugs is thought to result either from the binding of the drug to the interface enzyme– substrate or from a down-regulation of the G-protein-coupled PLA2 signaling (Myers et al., 2001). It is, however, not clear whether and how far the reduction of PLA2 activity by antipsychotic drugs is related to their clinical effects in schizophrenia. We speculate that increased PLA2 activity may be an inborn metabolic trait that affects brain maturation, increasing the risk for psychosis in a subgroup of schizophrenic patients. Accordingly, we would not expect a short-term effect of the reduction of the enzyme activity upon psychopathology. Moreover, we recently reported that the short-term inhibition of phospholipid metabolism by neuroleptics disappears after the continuation of treatment for over 6 months (Schmitt et al., 2001). Our finding of a relationship between increased PLA2 activity and absent response to niacin supports the assumption that absent response does reflect a disordered phospholipid metabolism in schizophrenia. The higher inhibition of PLA2 in our patients who turned responsive to niacin after drug treatment is in line with Glen et al. (1996), who reported on increased membrane arachidonic acid content in schizophrenic patients that converted from absent to positive niacin response after treatment. Taken together, the present results support the findings of a disrupted phospholipid metabolism in a subgroup of schizophrenic patients, and suggest that an absent response to the niacin test may be useful to identify these patients, for instance, for future trials testing the efficacy of alternative treatments aiming to restore the phospholipid metabolism in schizophrenia (Peet et al., 2001).
Acknowledgements This study is supported by the Fundacßa˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) grant 97/ 11083-0. The Laboratory of Neuroscience receives financial support from the Associacßa˜o Beneficente Alzira Denise Hertzog da Silva (ABADHS).
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References Albers, M., Meurer, H., Marki, F., Klotz, J., 1993. Phospholipase A2 activity in serum of neuroleptic-naive psychiatric in patients. Pharmacopsychiatry 26, 94 – 98. American Psychiatric Association, 1994. Diagnostic and Statistical Manual of Mental Disorders, 4th edn. American Psychiatric Press, Washington, DC. Easton, T., Hirsch, S.R., Das, I., Kidane, L., Richardson, A.J., Puri, B.K., 2000. The relationship of the area under the niacin flush dose – response curve and schizophrenia status. Schizophr. Res. 41, 244. Fiedler, P., Wolkin, A., Rotrosen, J., 1986. Niacin-induced flush as a measure of prostaglandin activity in alcoholics and schizophrenics. Biol. Psychiatry 21, 1347 – 1350. Gattaz, W.F., Kollish, M., Thurn, T., Virtanen, J.A., Kinnunen, P.K., 1987. Increased plasma phospholipase A2 activity in schizophrenic patients: reduction after neuroleptic therapy. Biol. Psychiatry 22, 421 – 426. Gattaz, W.F., Hubner, C.V.K., Nevalainen, T.J., 1990. Increased serum phospholipase A2 activity in schizophrenia: a replication study. Biol. Psychiatry 28, 495 – 501. Gattaz, W.F., Schmitt, A., Maras, A., 1995. Increased platelet phospholipase A2 activity in schizophrenia. Schizophr. Res. 16, 1 – 6. Glen, A.I., Cooper, S.J., Rybakowski, J., Vaddadi, K., Brayshaw, N., Horrobin, D.F., 1996. Membrane fatty acids, niacin flushing and clinical parameters. Prostaglandins Leukot. Essent. Fat. Acids 55, 9 – 15. Horrobin, D.F., 1980. Niacin flushing, prostaglandin E and evening primrose oil: a possible objective test for monitoring therapy in schizophrenia. Orthomol. Psychiatry 9, 33 – 34. Kay, S.R., Fiszbein, A., Opler, L.A., 1987. The positive and negative symptom scale for schizophrenia. Schizophr. Bull. 13, 261 – 276. Morrow, J.D., Awad, J.A., Oates, J.A., Roberts, L.J., 1992. Identification of skin as a major site of prostaglandin D2 release following oral administration of niacin in humans. J. Invest. Dermatol. 98, 812 – 815. Murrell, T.W., Taylor, W.M., 1959. The cutaneous reaction to nicotinic acid (niacin)-furfuryl. Am. Med. Assoc. Arch. Dermatol. 79, 93 – 98. Myers, C.S., Contreras, M.A., Chang, C.C.J., Rapoport, S.I., Appel, N.M., 2001. Haloperidol downregulates phospholipase A2 signaling in rat basal ganglia circuits. Brain Res. 896, 96 – 101. Peet, M., Brind, J., Ramchand, C.N., Shah, S., Vankar, G.T., 2001. The double-blind placebo-controlled study of eicosapentaenoic acid in the treatment of schizophrenia. Schizophr. Res. 49 (3), 243 – 251. Puri, B., Richardson, A.J., Easton, T., 2000. Association of niacin flush response with schizophrenia symptoms. Schizophr. Res. 41, 251. Ross, B.M., Hudson, C., Erlich, J., Warsh, J.J., Kish, S.J., 1997. Increased phospholipid breakdown in schizophrenia: evidence for the involvement of a calcium-independent phospholipase A2. Arch. Gen. Psychiatry 54 (5), 487 – 494. Rybakowski, J., Weterle, R., 1991. Niacin test in schizophrenia and affective illness. Biol. Psychiatry 29, 834 – 836.
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Schmitt, A., Maras, A., Gattaz, W.F., 2001. Effects of antipsychotics treatment on membrane phospholipid metabolism in schizophrenia. J. Neural Transm. 180 (8 – 9), 1081 – 1091. Shah, S.H., Vankar, G.K., Peet, M., Ramchand, C.N., 2000. Unmedicated schizophrenic patients have a reduced niacin skin flush in response to topical niacin. Schizophr. Res. 41, 251. Thuren, T., Virtanen, J.A., Lalla, M., Kinnunen, P.K., 1985. Fluorometric assay for phospholipase A2 in serum. Clin. Chem. 31, 714 – 717.
Trzeciak, H.I., Kalancinski, W., Mlecki, A., Kokot, D., 1995. Effect of neuroleptics on phospholipase A2 activity in the brain of rats. Eur. Arch. Psychiatry Clin. Neurosci. 245 (3), 179 – 182. Ward, P.E., Glen, A.I.M., 2001. Niacin response and phospholipids in psychiatry. World J. Biol. Psychiatry 2 (Suppl. 1), 296. Ward, P.E., Sutherland, J., Glen, E.M.T., Glen, A.I.M., 1998. Niacin skin flush in schizophrenia: a preliminary report. Schizophr. Res. 29, 269 – 274.
Increased phospholipase A2 activity in schizophrenia with absent response to niacin Hildeberto Tavares Jr., Juliana Yacubian, Leda L. Talib, Na´dia R. Barbosa, Wagner F. Gattaz* Laboratory of Neuroscience, Department and Institute of Psychiatry, Faculty of Medicine, University of Sa˜o Paulo, Rua Dr. Ovı´dio Pires de Campos s/n, P.O. Box 3671, 05403-010 Sa˜o Paulo-SP, Brazil Received 16 November 2001; received in revised form 8 February 2002; accepted 14 February 2002
Abstract An absent response to the niacin skin test has been reported to occur in about 80% of schizophrenic patients, as compared to 20% of healthy individuals. Niacin provokes redness in skin caused by a capillary vasodilatation mediated by prostaglandins. The metabolism of prostaglandins is regulated by the enzyme phospholipase A2 (PLA2). Several studies have reported increased PLA2 activity in schizophrenia. In this study we investigated the relationship between niacin response and PLA2 activity in 38 drug-free schizophrenic patients and in 28 healthy controls. Twenty-two of these patients were reevaluated after 8 weeks under treatment with new generation antipsychotic drugs. Niacin response was absent in 23% of the schizophrenic patients and in 14% in controls (n.s.). PLA2 activity was higher in schizophrenics than in controls (344 F 115 vs. 290 F 71 pmol/ml/min; p = 0.03). Patients with absent response to niacin had the highest PLA2 activity as compared to those with positive response (426 F 155 vs. 319 F 111; p = 0.02). After 8 weeks on antipsychotic treatment, PLA2 activity was reduced (355 F 115 before, 267 F 39 after, p = 0.001) and 4 out of 13 patients with absent response to niacin converted to positive. The reduction of PLA2 activity in these patients was higher than in patients who remained with absent response (36% vs. 23%). Our data support the findings that absent response to niacin is more frequent in schizophrenic than in healthy individuals although the magnitude of the difference was smaller than that reported in the literature. The relationship between absent response to niacin in schizophrenia and increased PLA2 activity suggests further that the skin test may be useful to easily identify a subgroup of patients with a disordered phospholipid metabolism. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Niacin test; Phospholipase A2 activity; Schizophrenia; Antipsychotic drugs
1. Introduction Niacin is a water-soluble vitamin B that causes skin redness when administrated orally or topically to individuals (Murrell and Taylor, 1959). This response is secondary to the liberation of prostaglandin D2 by skin *
Corresponding author. E-mail address: [email protected] (W.F. Gattaz).
macrophages (Morrow et al., 1992). Decreased or absent response to oral niacin has been reported in schizophrenic patients (Horrobin, 1980; Fiedler et al., 1986; Rybakowski and Weterle, 1991). Ward et al. (1998) found 83% of nonresponse to cutaneous administration of niacin (aqueous methyl nicotinate) vs. 23% in healthy controls. Such results have been confirmed by other groups (Easton et al., 2000; Shah et al., 2000), suggesting that niacin response could indicate a bio-
0920-9964/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0920-9964(02)00281-5
2
H. Tavares Jr. et al. / Schizophrenia Research 61 (2003) 1–6
logically more homogeneous subgroup of schizophrenic patients. Phospholipase A2 (PLA2) is an enzyme that cleaves fatty acids from the sn2-position of phospholipids in cellular membranes, liberating free fatty acids and lisophospholipids. Arachidonic acid (AA) is produced in this process and generates prostaglandins by the action of cyclo-oxygenase. Increased PLA2 activity has been found in serum, plasma, platelets and brain tissue from schizophrenic patients (Gattaz et al., 1987, 1990, 1995; Ross et al., 1997), although negative results were also reported by one study (Albers et al., 1993). In face of the modulation of prostaglandin synthesis by PLA2, we investigated, in the present study, the relationship between the enzyme activity and niacin response in schizophrenia.
2. Material and methods 2.1. Subjects and clinical assessment The study was performed at the Department and Institute of Psychiatry of the University of Sa˜o Paulo. The sample comprises of 38 schizophrenic patients (age 32.5 F 10.3 years; 19 male, 19 female) and 28 healthy individuals (age 30.5 F 8.2 years; 11 male, 17 female). All individuals were included in the study after having signed an informed consent. To be included in the study, subjects should be between 18 and 60 years old and should not have taken any analgesic or antiinflammatory drugs the last 2 days. Individuals with diabetes, rheumatological or dermatological diseases were not included in the study. Controls were not under any psychotropic medication and could not have any first degree relative with schizophrenia. Schizophrenic patients were diagnosed using the Structural Clinical Interview for DSM-IV (American Psychiatric Association, 1994) and should score at least 60 in the Positive and Negative Symptoms Scale for Schizophrenia— PANSS (Kay et al., 1987). All patients (n = 38) were from the community and had been ill for 7.8 F 6.8 years and had been previously hospitalized 2.3 F 1.3 times; all of them were drug-free for at least 2 weeks (mean 43.6 F 62.6 weeks) and 19 were drug naı¨ve. The mean total PANSS score for the sample was 82.4 F 20.0 and the sub-scores were 20.1 F 4.8 for positive symptoms; 27.2 F 9.9 for neg-
ative symptoms and 34.7 F 10.9 for general psychopathology. Twenty-two of the schizophrenic patients were reevaluated after 8 weeks of treatment with new generation antipsychotics (14 with olanzapine mean daily doses of 9.5 F 7.5 mg, 6 with amisulpride 500 F 300 mg and 2 with risperidone 6 mg). 2.2. Niacin test Niacin skin test was performed applying solutions of aqueous methyl nicotinate (0.1, 0.01, 0.001 and 0.0001 M) to four corresponding pieces of filter paper (2 2 cm) attached to the forearm skin with a nonallergic adhesive plastic strip. The strip was let in contact with the skin for 5 min and the response was evaluated every 5 min from 5 to 30 min, without washing niacin solution from the skin. The flush response was then scored as 0 = no erythema, 1 = incomplete erythema, 2 = complete erythema or 3 = erythema plus edema (Ward et al., 1998). Scores 0 and 1 were considered absent response and scores 2 and 3, positive. 2.3. PLA2 determination Serum was obtained from 10-ml blood collected in glass tubes without additions, kept at room temperature. It was immediately centrifuged in 3000 rpm for 15 min. Aliquots of 30 Al were frozen ( 70 jC). PLA2 activity in serum was then determined by a fluorimetric assay based on the procedure used by Thuren et al. (1985), with the modifications described bellow: 2.3.1. Chemicals 1-Octacosanyl-2-(1-pyrenehexanoyl)-sn-glycero-3phosphomethanol ammonium salt (C28-O-PHPM) and (pyren-1-yl) hexanoic acid (Molecular Probes, USA); n-heptan, toluene; ethanol; methanol; chloroform; hydrogen chloride and potassium carbonate (Merck, Germany); Trizma type pH 7.4 (Sigma USA); sodium borate (Reagen, Brazil); all chemicals were of analytical grade. 2.3.2. Preparation of substrate Dilute the C28-O-PHPM in toluene/ethanol (1:2, v/v) to obtain a concentration of 280 nmol/ml (stock solution). Evaporate 225 nmol/ml of this solution under a gentle stream of nitrogen and dissolve the residue in 800
H. Tavares Jr. et al. / Schizophrenia Research 61 (2003) 1–6
Al of ethanol (ethanolic solution). Rapidly add this solution into a test tube containing 5.6 ml of Tris (hydroxymethyl) aminomethane (Tris) – hydrogen chloride buffer (20 mmol/l, pH 7.4) (working solution). Keep the C28-O-PHPM solution on ice. 2.3.3. Assay procedure Add into a glass test tube 160-Al working solution of substrate and 40-Al serum, using as blank Tris – hydrogen chloride buffer (20 mmol/l, pH 7.4). Vortex both thoroughly and incubate at 37 jC in the dark in a shaking water bath for 60 min. Stop the reaction by adding 1.3 ml chloroform/methanol: n-heptan, (1.25:1.41:1, v/v/v) and vortex thoroughly for 30 s. Add 300-Al sodium borate– potassium carbonate buffer (0.14 mol/l each, pH 10.5) and thoroughly mix the solution. Centrifuge the tubes for 10 min at 2000 g at room temperature. Pipette 750 Al of the aqueous phase into a magnetically stirred four-window quartz cuvette and add 750-Al 40% methanol solution. Set fluorescence excitation at 343 nm and measure emission at 400 nm. 2.3.4. Instrument calibration Adjust the fluorescence intensity to 1 106 readings/s when the cuvette contains 50 pmol of (pyren-1yl) hexanoic acid in 40% methanol solution and 750 Al of the aqueous phase of control serum (volunteer)— final volume: 1.5 ml. We measured fluorescence with a fluorescence spectrometer with two emission subsystems (Photon Technology International—Canada) and for the entrance and exit sides of both the excitation and emission monochromators (2.5 turns of the slit micrometers—bandpass 5 nm) and excitation wavelength: 343 nm and emission wavelength: 400 nm, for both channels. PLA2 activity is expressed as picomoles of free (pyren-1-yl) hexanoic acid produced per minute per milliliter of serum and is calculated as follows: PLA2 activity ðpmol min1 ml1 Þ ¼
I 25 1:33 1:54 0:00005 60 0:040
where: I = fluorescence intensity, in arbitrary units read from spectrofluorometer; 25 = correction factor to obtain the activity per milliliter of serum; 1.33 = adjust-
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ment to whole aqueous phase; 1.54 = recovery of the substrate is estimated as 65%; 60 = incubation time (min); 0.040 = volume of serum (ml); 0.00005 = conversion of the fluorescence intensity reading into picomoles of (pyren-1-yl) hexanoic acid. Statistical analysis was performed with chi-square test for the analyses of niacin categorical data and two-tailed t test for PLA2 activity.
3. Results Absent response to topic niacin in schizophrenia was more frequent than in controls for most of the concentrations and measurement times (Table 1). However, a trend for significance was found only for the concentration of 0.001 M at 10, 25 and 30 min. To allow comparisons with other reports that used the same methodology as in the present study, we will analyze the data related to the niacin concentration of 0.01 M at 10 min (Ward et al., 1998). Although the control individuals and the schizophrenic patients were not matched by gender, no significant statistical differences were found in niacin test comparing the groups separated by sex. We also did not find differences in niacin test between the previously medicated (n = 19) and the drug naı¨ve patients (n = 19). No significant differences in psychopathological scores and clinical improvement were found between
Table 1 Absent response to niacin (%) in schizophrenic patients and in healthy controls
0.01 M 5 min 0.01 M 10 min 0.01 M 15 min 0.01 M 20 min 0.01 M 25 min 0.01 M 30 min 0.001 M 5 min 0.001 M 10 min 0.001 M 15 min 0.001 M 20 min 0.001 M 25 min 0.001 M 30 min
Schizophrenia (n = 38) (%)
Control (n = 28) (%)
p
Chi
60 23.7 10.5 10.5 18.4 18.4 100 86.8 84.2 84.2 84.2 86.8
57.1 14.2 14.2 14.2 17.2 17.2 96.4 67.8 67.8 67.8 64.2 64.2
0.49 0.26 0.46 0.46 0.54 0.54 0.62 0.06 0.10 0.10 0.06 0.03
0.08 0.9 0.21 0.21 0.05 0.05 0.05 3.48 2.46 2.46 3.48 4.67
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Table 2 PANSS scores in 22 schizophrenic patients according to the response (positive or absent) after 10 min of niacin 0.01 M
Total PANSS Positive PANSS Negative PANSS
Absent niacin (n = 9)
Positive niacin (n = 13)
p
t
90.8 F 28.2 19.0 F 4.8 30.8 F 8.7
80.0 F 17.3 20.4 F 4.9 26.1 F 10.2
0.1 0.86 0.71
0.82 0.57 1.01
Table 4 PLA2 activity in 38 schizophrenic patients compared to 28 controls; in 22 schizophrenic patients before and after 8 weeks of treatment with new generation antipsychotics; and in 9 schizophrenic patients with absent response to niacin compared to 13 patients with positive response to niacin (after 10 min of niacin 0.01 M) Schizophrenia (n = 38)
Control (n = 28)
p
t
344 F 115
290 F 71
0.03
2.18
Before Treatment (n = 22) After Treatment (n = 22)
patients with absent and positive response to niacin, using t test for comparison of means (Table 2), in any niacin concentration or time of evaluation. After 8 weeks on antipsychotic drug treatment, 4 of the 13 patients who were at baseline were niacin-negative turned into positive ( p = 0.06, Table 3). PLA2 activity (Table 4) was increased in schizophrenics as compared to controls (344 F 115 vs. 290 F 71 pmol/ml/min; p = 0.03; t = 2.18). PLA2 activity decreased by 25% in the 22 patients reevaluated after 8 weeks on antipsychotic treatment (355 F 11 before and 267 F 39 after, p = 0.001; t = 4.04). No correlations were found between PLA2 activity at baseline nor PLA2 reduction after treatment with psychopathological scores and improvement. There was not any correlation between PLA2 activity and the drug-free period (Spearman = 0.10, p = 0.57). Patients with absent response to niacin (n = 13) had higher PLA2 activity than those with positive (n = 9) response (426 F 155 vs. 319 F 111, p = 0.02; t = 2.3).
Table 3 Absent response to niacin (%) at different times and concentrations in 22 schizophrenic patients before and after 8 weeks on antipsychotic drug treatment
0.01 M 5 min 0.01 M 10 min 0.01 M 15 min 0.01 M 20 min 0.01 M 25 min 0.01 M 30 min 0.001 M 5 min 0.001 M 10 min 0.001 M 15 min 0.001 M 20 min 0.001 M 25 min 0.001 M 30 min
Before treatment (%)
After treatment (%)
p
Chi
60.0 22.7 4.5 9.0 13.6 13.6 86.3 86.3 81.8 81.8 77.3 81.8
40.9 4.5 4.5 9.0 13.6 13.6 77.2 77.2 68.2 68.2 68.2 68.2
0.03 0.06 0.90 0.17 0.04 0.03 0.12 0.12 0.07 0.07 0.16 0.07
5.5 5.8 4.2 4.4 8.3 8.2 3.8 3.8 4.2 4.2 2.37 4.2
355 F 11
267 F 39
Absent Niacin (n = 9)
Positive Niacin (n = 13)
426 F 155
319 F 111
0.001 4.04
0.02
2.3
The patients who converted niacin response (0.01 M after 5 min) from absent into positive after treatment (n = 4) had a 36% reduction in PLA2 activity, against 23% in those who remained nonresponsive (n = 5).
4. Discussion Absent flushing in response to niacin was more frequent in schizophrenic patients than in controls in our sample, but the magnitude of the difference was less than that reported by other groups. Using a similar niacin test as in our study, for the concentration of 0.01 M, Ward et al. (1998) found 83% absent response in drug-treated schizophrenic patients and 23% in controls. Using a quantitative analysis, Shah et al. (2001) found less skin flushing in schizophrenic patients. We are not able to explain these differences among the results, as similar test procedures were used in those studies. Puri et al. (2000) found a predominance of negative symptoms in schizophrenic patients with absent response to niacin. In our sample, we could not replicate this relationship between niacin response and psychopathology scores (Table 2), which was also not observed in other studies (Glen et al., 1996; Ward and Glen, 2001). In line with previous studies (Gattaz et al., 1987; Ross et al., 1997), PLA2 activity in serum was increased in drug-free schizophrenia and the enzyme activity was reduced after antipsychotic drug treatment. It has already been shown that haloperidol (Gattaz et al., 1987) and other first generation anti-
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psychotics (Trzeciak et al., 1995) reduce PLA2 activity. The present results extend those findings also to new generation antipsychotics such as olanzapine, amisulpride and risperidone. Reduced PLA2 activity trough the action of antipsychotic drugs is thought to result either from the binding of the drug to the interface enzyme– substrate or from a down-regulation of the G-protein-coupled PLA2 signaling (Myers et al., 2001). It is, however, not clear whether and how far the reduction of PLA2 activity by antipsychotic drugs is related to their clinical effects in schizophrenia. We speculate that increased PLA2 activity may be an inborn metabolic trait that affects brain maturation, increasing the risk for psychosis in a subgroup of schizophrenic patients. Accordingly, we would not expect a short-term effect of the reduction of the enzyme activity upon psychopathology. Moreover, we recently reported that the short-term inhibition of phospholipid metabolism by neuroleptics disappears after the continuation of treatment for over 6 months (Schmitt et al., 2001). Our finding of a relationship between increased PLA2 activity and absent response to niacin supports the assumption that absent response does reflect a disordered phospholipid metabolism in schizophrenia. The higher inhibition of PLA2 in our patients who turned responsive to niacin after drug treatment is in line with Glen et al. (1996), who reported on increased membrane arachidonic acid content in schizophrenic patients that converted from absent to positive niacin response after treatment. Taken together, the present results support the findings of a disrupted phospholipid metabolism in a subgroup of schizophrenic patients, and suggest that an absent response to the niacin test may be useful to identify these patients, for instance, for future trials testing the efficacy of alternative treatments aiming to restore the phospholipid metabolism in schizophrenia (Peet et al., 2001).
Acknowledgements This study is supported by the Fundacßa˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) grant 97/ 11083-0. The Laboratory of Neuroscience receives financial support from the Associacßa˜o Beneficente Alzira Denise Hertzog da Silva (ABADHS).
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