Plasma membrane phospholipid and cholesterol distribution of skin fibroblasts from drug-naive patients at the onset of psychosis

SCHIZOPHRENIA RESEARCH Schizophrenia Research 13 (1994) 239-247

Plasma membrane phospholipid and cholesterol distribution of skin fibroblasts from drug-naive patients at the onset of psychosis Sahebarao P. Mahadik a,*, Sukdeb Mukherjee a, Elizabeth E. Correnti b, Hemant S. Kelkar a, Chandramohan G. Wakade a, Richard M. Costa a, R. Scheffer b a Department

of Psychiatry and Health Behavior, Medical College of Georgia, Augusta, GA 30912, USA, ‘** Department of Veterans Affairs Medical Center (151-D), Augusta, GA 30904-6285, USA, b D.D. Eisenhower Army Medical Center, Fort Gordon, GA, USA Received

29 October

1993; accepted

22 April 1994

Abstract Contents of plasma membrane major phospholipids, cholesterol, and cholesteryl esters of fibroblasts from drugnaive psychotic patients were compared with those from normal controls. Total membrane lipids were extracted and individual lipids were separated on high-performance thin-layer chromatography. The contents of lipid bands were quantitated by densitometric scanning and comparing with standards. Contents of total phospholipids as well as phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine were significantly lower in fibroblasts from patients than in those from normal controls (P
Psychosis;

Plasma membrane

phospholipid;

Cholesterol;

1. Introduction Abnormal composition of lipids and esterified fatty acids has been reported in plasma (Horrobin et al., 1989; Kaiya et al., 1991), red blood cells (Stevens, 1972; Hitzeman et al., 1984; Glen et al., 1994; Yao et al., 1993, this issue), and post-mortem frontal cerebral cortical tissue (Horrobin et al., 199 1) of schizophrenic patients. Elevated levels of adrenic (22:4n - 6) and docosapentaenoic (22% - 3) acids have been found in plasma from twins (concordant > discordant for schizophrenia)

* Corresponding 706-823-3977.

author. Tel.: 706-733&-0188, ext. 2670; Fax:

0920-9964/94/$7.00 0 1994 Elsevier Science B.V. All rights reserved SSDZ 0920-9964(94)00026-5

Fibroblast

(Bates et al., 1992). Furthermore, in concordant twins, the levels of linoleic acid were lower, and the levels of y-linolenic acid and eicosapentaenoic acid were significantly higher. A role of abnormal levels of metabolites of released plasma membrane fatty acids (eicosanoids and prostaglandins) in schizophrenia has been suggested (Abdulla and Hamadah, 1975; Feldberg, 1976; Van Kammen et al., 1989; Horrobin, 1990). The alterations in fatty acids are consistent with the significantly higher levels of phospholipase A, in serum (Gattaz et al., 1990; Rotrosen et al., 1993, personal communication), and platelet membranes (Gattaz et al., 1993; personal communication), and with increased levels of platelet lysophosphatidylcholine of schizophrenic patients (Pangerl et al., 1991).

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More recently, abnormal levels of red blood cell (RBC) fatty acids have been found in schizophrenic patients with predominantly negative symptoms, but not in those without such symptoms (Horrobin et al., 1993; Glen et al., 1994). The findings of those studies are consistent with disordered phospholipid metabolism in schizophrenia (Rotrosen and Wolkin, 1987). 31P-NMR spectroscopy has shown decreased levels of phosphomonoesters (PME) and increased levels of phosphodiesters (PDE), indicative of altered neural membrane lipid metabolism, in the prefrontal cortex of schizophrenic patients (Pettegrew et al., 1991; Stanley et al., 1993; this issue) as well as in one subject, considered to be normal at the time of study, who subsequently developed schizophrenia implicating a role of membrane pathoogy in the pathogenetic process 1991). of schizophrenia (Keshavan et al., Decreased levels of PME, but no change in PDE, was reported in chronic, medicated schizophrenic patients (Williamson et al., 1991; Stanley et al., 1993; this issue). The alterations in lipid and fatty acid distribution in RBCs from schizophrenic patients may provide clues about the disordered lipid metabolism. However, past investigations have led to disparate findings, which may be the result of confounding effects of dietary variability, age, sex, metabolic state, disease process and its treatment (Kuksis, 1978; Dougherty et al., 1987). Also, both in vivo analysis of brain lipids and in vitro analysis of RBCs are not suitable systems for studies of dynamic regulatory molecular processes underlying altered lipid and fatty acid metabolism. Such information is critical for developing rational strategies to correct the changes in membrane lipid or fatty acid distribution in order to modify the course of the disease or the treatment response of patients. Several experimental systems can be used to investigate the possible regulatory molecular processes of abnormal lipid metabolism in schizophrenia. These include: (a) Neuronal cultures from biopsied or post-mortem brains of schizophrenic patients - this is in the early stages of development and such cells are difficult to grow in sufficient quantities. (b) Neuronal cultures from aborted fetuses of schizophrenic mothers (Freedman et al.,

Research I3 (1994) 239-247

1992) - their use is also limited by availability of such tissue and limited neuronal survival in culture. (c) Skin fibroblasts these are easier to grow and maintain in culture for considerable lengths of time, and can be grown in large quantities without known phenotypic alterations (Edelstein and Breakfield, 1980). Since lipid abnormalities may be present in both neural and non-neural tissues, molecular mechanisms underlying abnormal lipid metabolism may be common to all tissues. We have reported that skin fibroblasts from schizophrenic patients show abnormal growth and morphology when compared with those from normal subjects (Mahadik et al., 1991). We have further found that cell adhesiveness, surface distribution of fibronectin, and the growth response to basic fibroblast growth factor are altered in fibroblasts from patient (Wakade et al., 1993; Mahadik et al., 1994). These and the reported abnormal tyrosine transport in fibroblasts from schizophrenic patients (Hagenfeldt et al., 1987; Weisel et al., 1991) suggest that alterations in plasma membrane structure and function may be a result of the altered contents and distribution of membrane lipids and their fatty acids, since they play critical roles in these processes. In a preliminary study, we found that the membrane phospholipid distribution was abnormal in fibroblasts from chronic schizophrenic patients (Kelkar et al., 1993). Although fibroblasts in culture are considered to be free from the effects of medication as well as clinical state, there is always a concern regarding their long-lasting effects on cell phenotype. All these studies further suggest that fibroblasts from drug-naive, first-episode psychotic patients may be preferable for investigating dynamic regulatory molecular processes underlying the altered lipid metabolism relin schizophrenia. We have established fibroblast cell lines from skin biopsies of drug-naive patients at the onset of psychosis and reported that these cells show abnormal growth and morphology when compared with fibroblasts from normal controls (Mahadik et al., 1993a; Mukherjee et al., 1993). We report here on the abnormal plasma membrane lipid distribution in these fibroblasts.

S. P. Mahadik et al./Schizophrenia

2. Methods

Research 13 (1994) 239-247

membranes membrane

(Lange fractions

et al., 1989) we used for these analyses.

241

total

2.1. Subjects The sample comprised 10 patients admitted at the D.D.E. Army Medical Center, Fort Gordon, GA for treatment of their first episode of psychosis and six normal control subjects. DSM-IIIR diagnoses were based on structured clinical interviews, review of records, and interview with a parent by an AMC psychiatrist. Four patients met criteria for a diagnosis of schizophrenia, and six were classified as having schizophreniform disorder since they had not yet met the duration criterion for a diagnosis of schizophrenia. All subjects were medically healthy, and none had a history of substance abuse, severe head injury, or seizure disorder. Additionally, normal controls had no family history of psychosis. Although patients were younger than the normal controls (mean &-SD = 21.2k2.75 yrs, and 28.17+ 1.83 yrs, respectively, P ~0.05) all subjects were younger than 30 yrs of age. The average duration of illness of 4.6 days. There was no significant difference between patients and normal controls in gender distribution or ethnic background. All subjects gave informed consent for participation in the research. 2.2. Fibroblust cultures Skin fibroblast cultures were established using procedures that have been described in detail previously (Mahadik et al., 1991). All the cultures were stored at - 150°C or in liquid nitrogen between passages 4 to 8, and revived and grown for this experiment in flasks until they attained 80% confluency or grown to the same time point. Cells were detached by quick trypsinization and collected by centrifugation. After washing cells with ethylene glycol tetra-acetic acid (EGTA)phosphate buffer, cells were suspended in water containing 1 mM EGTA and lysed by homogenization and freeze-thawing. The EGTA was used to inhibit Ca 2+-dependent lip ases. Membranes were collected by centrifugation and used fresh as a source of total lipids. Since over 90% of the cholesterol and cholesteryl esters and over 50% of phospholipids in fibroblasts are present in plasma

2.3. Total membrane lipid extraction and separation of phospholipids The procedures used have been described in detail previously (Mahadik et al., 1989, 1993b). Total lipids were extracted by two treatments of plasma membrane fractions with 10 vol. of chloroform:methanol (C:M, 2:l v/v). Both extracts were combined and dried under nitrogen with care to protect the sensitive lipids from oxidation. The concentrate was dissolved in C:M and partitioned by adding a 20% volume of water (Folch et al., 1957). The lower phase containing neutral lipids, glycolipids, and phospholipids was processed through a unisil (silicic acid) column for separation (Rouser et al., 1967). The column was washed with chloroform to elute the neutral lipid fraction (cholesterol, free fatty acids and triglycerides), with methanol containing 10% acetone to elute glycolipids, and finally with methanol to elute phospholipids. Each of these fractions was collected with care, dried, and stored over desiccant under nitrogen until used. The separation and quantitation of individual phospholipids from respective fractions were done by separating on high performance TLC followed by densitometric scanning. 2.4. Separation und quantitation of individual phospholipids Separation and quantitation of major individual phospholipid classes (phosphatidylethanolamine, PE; phosphatidylcholine, PC; phosphatidylserine, PS; phosphatidylinositol, PI; phosphatidylglycerol, PG), cholesterol and cholesteryl esters were done as described by Kates (1986). Separation was carried out using high performance thin-layer chromatographic (HPTLC) plates of silica gel 60 (0.2 mm; 10 x 10 cm; Whatman) activated at 100°C for 15 min. Each plate was developed with C/M/acetic acid/water, 60:50: 1:4 (v/v) and dried. Phospholipid bands were stained with phosphorus specific reagent, molybdenum blue (Sigma). Intense blue bands develop within 15 min. Lipid bands were visualized by heating in an oven at

S. P. Mahadik et al./Schizophrenia Research 13 (1994) 239-247

242

110°C for 30 min. The identity of individual phospholipids was established by comparing the migration of commercially available standards. The TLC chromatograms were scanned with a Shimadzu CS-910 TLC scanner, using absorption densitometry (475 nm) in reflection mode at 20 mm/min. Each sample was run in duplicate and each lane was scanned twice. The data were analyzed by integration with a Shimadzu CR-IA data processor. No value was included unless it exceeded 300 PV per second. The absorbance yields of known individual phospholipid species were used to determine their content in membranes, which was expressed as ,ug/mg membrane protein. The replicate analyses of each sample on different HPTLC plates differed by less than 7%.

3. Results 3.1. Phospholipid distribution in membranes of jibroblasts from schizophrenic patients As shown in Table 1, the total membrane phospholipid content was significantly lower in fibroblasts from the patients as compared with normal controls (P PI > PE. There was no change in the content of PC. These changes were not related to sex or age. 3.2. Cholesterol and cholesteryl esters in membranes ofjibroblasts from schizophrenic patients

As shown in Table 2, the content of the total cholesterol fraction of plasma membrane was Table 1 Contents= of plasma membranes phospholipids of psychosis and normal controls PC

significantly lower in fibroblasts patients than in fibroblasts from normal controls (P
4. Discussion These findings indicate that levels of both phospholipid and cholesteryl esters are different in membrane fractions (pg:mg membrane protein) of cultured skin fibroblasts from drug-naive patients at the first onset of psychosis as compared with normal controls. The lower membrane total phospholipid:protein ratios were owing to significantly lower levels of three of the four major membrane phospholipids: PS, PI, and PE. There was no change in the level of the major phospholipid PC, which comprises about 45% of the total membrane phopsholipids. These differences between patients and normals were not related to age or sex. Identical membrane Table 2 Contents6 of plasma membranes cholesterol esters of fibroblasts form drug-naive patients of psychosis and normal controls esters

and cholesteryl at the first onset

Cholesterol

Cholesteryl

Total cholesterol

Normals

317.9+_ 153.21

324.63k95.44

642.53 +_206.92

Patients

249.72k2.42b

150.02_f42.39d

399.75 * 89.54”

aAll

values are means* SD, expressed as ng lipid/mg membrane protein. Statistical analyses were done by using 2-tailed unpaired ttests: bns; ‘P
(PC, PS, PI, PE and total)

PS

PI

of fibroblasts

from drug-naive

PE

patients

at the first onset

Total PL

40.92 k 14.43

181.18+10.22

27.09&-8.19’

123.39k27.33”

“All values are means+SD, expressed as pg lipid/mg membrane protein. PC, phosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PL, phospholipid. Statistical analyses were done by using 2-tailed unpaired r-tests: bns; ‘P
PS, phosphatidylserinc;

Normals

17.74+

15.6

35.38k8.51

26.67*

Patients

70.33+

14.1Sb

16.69+7.59d

14.75+5.17=

10.16

S. P. Mahadik et al./Schizophrenia

fatty acid levels have been found in blacks and whites under similar dietary conditions (Crawford and Sinclair, 1992). This makes it unlikely that race would influence the data, since fibroblasts were grown under identical conditions. The values for contents of phospholipids found in normal subjects in this study were similar to values reported previously for human fibroblasts (Karmiol and Bettger, 1988; Lange et al., 1989). Inconsistent findings have been reported on the phospholipid levels in erythrocytes. Early studies reported an increase in PS, and a decrease in PC and PE (Stevens, 1972; Hitzmann et al., 1984), but later studies failed to replicate these findings (Rotrosen and Wolkin, 1987; Rotrosen, personal communication). Large variability is also found in the amounts of esterified fatty acids in RBCs from schizophrenic patients, as well as normal controls from different countries (Manku et al., 1983). It has been reported that phospholipid and fatty acid levels in erythrocytes and plasma are influenced by diet, age, sex, metabolic state, and hormonal variations (Kuksis, 1978; Dougherty et al., 1987). This suggests that the disparate findings in RBC phospholipids may, at least in part, be due to the confounding effects of the above factors, as well differences across patients in clinical manifestations. The latter is consistent with the recent observation that an abnormal RBC fatty acid profile (decreased levels of unsaturated and increased levels of saturated fatty acids) in schizophrenic patients was associated with negative symptoms (Horrobin et al., 1993). The possibility that part of the altered phospholipid metabolism in RBCs is a result of treatment, is suggested by the findings of in vitro studies which have shown that exposure of human fibroblasts (Maziere et al., 1988) or mouse monocyte-macrophases (Houtia et al., 1988) to phenothiazines (e.g., trifluoperazine, chlorpromazine) increases the incorporation of fatty acids in membrane phospholipids and decreases the levels of cholesteryl esters. However, Yao et al. (1993, this issue) did not find differences in RBC fatty acids after > 5 weeks of drugfree period. Our preliminary studies on fibroblasts from established schizophrenic patients (Kelkar et al., 1993) and this study on fibroblasts from drug-

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naive first-episode psychotic patients support the notion that the abnormal lipid metabolism most likely predates the onset of manifest illness. This has been suggested previously based on altered levels of phospholipid metabolites (decreased PME, and increased PDE) in brain by 31P-NMR spectroscopy in first-episode, drug-naive schizophrenic patients (Pettegrew et al., 1991). Recently, lower levels of PE have been found in brain using the same methodology (Drs. MS. Keshavan and P. Williamson, personal communications, March 29, 1993). However, phospholipid analysis of postmortem schizophrenic brains did not show any change in the levels of phospholipids but rather showed significant changes in the distribution of fatty acid composition of PE (Horrobin et al., 1991). There was a significant increase in docosapentaenoic, no change in arachidonic, and significantly lower levels of linoleic, gamma-linolenic and dihomogamma-linolenic acids. Whereas, the levels of all of these polyunsaturated fatty acids were lower in red blood cells from schizophrenic patients with negative symptoms (Glen et al., 1994), and the levels of only n - 6 fatty acids were slightly lower and levels of n - 3 fatty acids were slightly higher in plasma (Horrobin et al., 1989). Some of these discrepancies between levels may reflect the complex and differential interactions of various factors, such as diet, on these tissues influencing differentially the fundamental defect in lipid metabolism. Regulation of lipid metabolism is poorly understood in vertebrate tissues, and even less in brain (Thomson, 1992). Based on the lower levels of polyunsaturated and higher levels of saturated fatty acids in RBCs in patients with negative symptoms, Glen et al. (1994) have suggested that there may be a defect in the transport of dietary EFAs (linoleic and gamma-linolenic acids) as well as incorporation of polyunsaturated fatty acids as a result of a possible defect in elongation and/or desaturation of dietary EFAs. Dietary supplementation of y1- 6 fatty acids in schizophrenic patients has been found to correct partly the levels of polyunsaturated fatty acids in red blood cells with concomitant improvements in psychiatric scores and memory (Vaddadi et al., 1989). This suggests that there may be a primary defect in membrane

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transport of dietary EFAs into the cells which can result in an increased availability of saturated fatty acids (locally synthesized), and thereby an increase in their incorporation (Yao et al., 1993, this issue). It is also important to point out that in vitro treatment of cultured cells by phenothiazines can increase the incorporation of both saturated and unsaturated fatty acids in membrane phospholipids (Houtia et al., 1988; Maziere et al., 1988). This is particularly important with respect to schizophrenic patients without negative symptoms who have normal levels of fatty acids in RBC membranes. It is likely that the normal levels of membrane fatty acids in this group of patients is a result of treatment. In other words, phenothiazines probably correct the defect in fatty acid incorporation into RBC membranes of patients with positive symptoms. The lower levels of PE observed in fibroblasts from drug-naive first-episode patients probably reflect defect in cellular transport of precursor EFAs into the cells as well as elongation, desaturation and or incorporation into lipids. PE, which constitues 38% of the total phospholipids in neuronal membrane (Suzuki, 1981) and about 24% in human fibroblasts (Karmiol and Bettger, 1988), is specifically enriched in polyunsaturated fatty acids compared to other phospholipids in the brain (Bernsohn and Cohen, 1972; O’Brien and Sampson, 1965). The levels of PE in tissue are determined either by intracellular levels of polyunsaturated fatty acids and their incorporation directly into PE or by direct exchange through the deacylation-acylation of fatty acids from other lipids (Thompson, 1992). The latter mechanism requires a proper balance between the phospholipase A, and fatty acid acyltransferases. The increased levels of phospholipase Aa, which have been found in plasma as well as in platelet membranes from schizophrenic patients (Gattaz et al., 1992; personal communication), can create a deficiency in polyunsaturated fatty acids and result in increased incorporation of saturated fatty acids. These studies further emphasize the usefulness of skin fibroblasts in culture for understanding better the molecular mechanisms of phospholipid metabolism in schizophrenia. Fibroblasts in culture can eliminate most of the confounding factors and

allow the identification of underlying regulatory molecular processes of lipid metabolism. However, the possibility of some irreversible phenotypic changes as a result of the disease state and its treatment can not be excluded. Ultimately, parallel studies on fibroblasts and RBCs from different sub-groups of patients may help to resolve some of these issues. Levels of cholesteryl esters were also found to be substantially lower in membrane fractions of fibroblasts from the patients, Significantly lower levels of cholesteryl esters have also been reported in the cerebral frontal cortex of schizophrenic patients (Horrobin et al., 1991). The role of cholesteryl esters in membrane function is unclear. They are known to be formed from excess cholesterol by esterification with fatty acids. However, their presence in membranes can affect membrane fluidity. In fibroblasts, over 90% of cholesterol and cholesteryl esters are located in plasma membranes (Lange et al., 1988) and over 50% is found in plasma membrane in brain (Suzuki, 1981). It is important to note that in vitro treatment of cultured cells by phenothiazines inhibit the enzyme A:cholesterol-0-acyltransferase, acylcoenzyme and reduce the levels of cholesteryl esters (Houtia et al., 1988; Maziere et al., 1988). This may, in part, explain the reduction in their levels in postmortem brains after years of treatment with phenothiazines. However, the lower levels of cholesteryl esters in fibroblasts of drug-naive patients probably reflect an underlying defect in fatty acid incorporation in cholesterol in schizophrenia. This supports the idea that in schizophrenia there is a defect in the regulation of fatty acid metabolism which is responsible for both phospholipid and, cholesterol esterification and therefore, for their reduced levels. It has been extensively documented using in vivo and in vitro systems that phospholipids play critical roles in almost every function of the cell membrane as well as its metabolic products in cellular functions (Bloj et al., 1973; Bourre et al., 1989). These include: ion and nutritional transport, receptor-mediated transfer of chemical signal transduction, generation and propagation of action potentials, and cell-cell interactions. There is also evidence that membrane changes can have

S. P. Mahadik et al.lSchizophrenia Research 13 (1994) 239-247

differential effects in different tissues of the body. The lower levels of PI and PE can influence signal transduction and processes mediated by second messengers. The significance of the phospholipid abnormalities observed in fibroblasts with respect to brain cell membrane function, if they are generalizable to brain cells, remain speculative. Certainly, alterations in membrane fatty acids and thereby membrane function of neural cells can have profound effects on brain development as well as brain function throughout life (Neuringer et al., 1986; Bourre et al., 1989; Crawford, 1992; Wainwright, 1992). If these lipid alterations in skin fibroblasts are generalized and if they were present during development and maturation, they could have profound and selective effects on brain development and maturation. Almost two-third of the brain dry weight is comprised of lipids which contain a very large proportion of polyunsaturated fatty acids compared to other organs (Suzuki, 1972; Sinclair, 1975), and these polyunsaturated fatty acids play a critical role in brain and behavioral development (Bernsohn and Cohen, 1972; Crawford, 1992; Wainwright, 1992). There is considreable evidence that neurodevelopmental problems contribute to the pathogenesis of schizophrenia (Fish et al., 1992; Weinberg et al., 1987; Mukherjee et al., 1992; Bloom, 1993).

5. Acknowledgments This research was supported in part by NIMH grants MH 41961, 46546 and 47002.

6. References Abdulla, Y.H. and Hamadah, K. (1975) Effects of ADP on PGE formation in blood platelets from patients with depression, mania and schizophrenia. Br. J. Psychiatry 127, 591-595. Bates, C., Horrobin, D.F. and Ells, K. (1992) Fatty acids in plasma phospholipids and cholesterol esters from identical twins concordant and discordant for schizophrenia. Schizophr. Res. 6, l-7. Bernsohn, J. and Cohen, S.R. (1972) Polyenoic fatty acid metabolism of phosphoglycerides in developing brain. In: Ciba Foundation Symposium, Elsevier, New York, pp. 159-178. Bloj, B., Morero, R.D. and Farias, R.N. (1973) Membrane

245

fluidity, cholesterol and allosteric transitions of membrane(Na+,K+)-ATPase and acetylchobound Mg ‘+-ATPase, linesterase from rat erythrocytes. FEBS Lett. 28, lOlllO5. Bloom, F. (1993) Comment: Advancing a neurodevelopmental origin for schizophrenia. Arch. Gen. Psychiatry 50, 2244227. Bourre, J.-M., Francois, L, Youyou, A., Dumont, O., Piciotti, M., Pascal, G. and Durand, G. (1989) The effects of dietary a-linolenic acid on the composition of nerve membranes, enzymatic activity, amplitude of electrophysiological parameters, resistance to poisons and performance of learning tasks in rats. J. Nutr. 119, 1880-1892. Crawford, M.A. (1992) Essential fatty acids and neurodevelopmental disorder. In: Bazan, N.G., et al. (Eds), Neurobiology of Essential Fatty acids, Plenum, New York, pp. 3077314. Crawford, M.A. and Sinclair, A.J. (1972) Nutritional influences in the evolution of mammalian brain. In: Ciba Foundation Symposium, Elsevier, New York, pp. 267-292. Dougherty, R.M., Galli, C., Ferro-Luzzi, A. and Iacono, J.M. (1987) Lipid and phospholipid fatty acid composition of plasma, red blood cells, and platelets and how they are affected by dietary lipids: a study of normal subjects from Italy, Finland, and the USA. Am. J. Clin. Nutr. 45, 443-455. Edelstein, S.B. and Breakenfield, X.0. (1980) Human fibroblast cultures. In: Hanin, I., Koslow, S.H. (Eds.), Physicochemical Methodologies and Psychiatric Research, Raven, New York, pp. 200-243. Feldberg, W. (1976) Possible association of schizophrenia with a disturbance in prostaglandin metabolism: A physiological hypothesis. Psychol. Med. 6, 359-369. Fish, B., Marcus, J., Hans, S.L,, Auerbach, J.G. and Perdue, S. (1992) Infants at risk for schizophrenia: Sequelae of a genetic neurointegrative defect. Arch. Gen. Psychiatry 49, 221-235. Folch, J., Lee, M. and Sloane-Stanley, G.H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 4977509. Freedman, R., Stromberg, I., Seiger, A. et al. (1992) Initial studies of embryonic transplants of human hippocampus and cerebral cortex derived from schizophrenic women. Biol. Psychiatry 32, 1148-l 163. Gattaz, W.F., Hubner, C.K., Nevalainen, T.J., Thuren, T. and Kinnunen, P.K.J. (1990) Increased serum phospholipase A, activity in schizophrenia: A replication study. Biol. Psychiatry 28, 4955501. Glen, A.I.M., Glen, E.M.T., Horrobin, D.F., Vaddadi, K.S., Spellman, M., Morse-Fisher, N., Ellis, K. and Skinner, F.K. (1994) A red cell membrane abnormality in a subgroup of schizophrenic patients: Evidence for two diseases. Schizophr. Res. 12, 53-61. Hagenfeldt, L., Venizelos. N., Bjerkenstedt, L. and Wiesel, F.A. (1987) Decreased tyrosine transport in fibroblasts from schizophrenic patients. Life Sci. 41, 274992757. Hitzemann, R., Hirschowitz, J. and Garver, D. (1984) Membrane abnormalities in the psychoses and affective disorders. Psychiatric Res. 18, 319-326. Horrobin, D.F., Manku, M.S., Morse-Fisher, N. and Vaddadi,

246

S. P. Mahadik et al. JSchizophrenia Research 13 (1994) 239-247

KS. ( 1989) Essential fatty acids in plasma phospholipids in schizophrenics. Biol. Psychiatry 25, 562-568. Horrobin, D.F., Manku, MS., Hillman, S., Iain, A. and Glen, M. (1991) Fatty acid levels in brains of schizophrenics and normal controls. Biol. Psychiatry 30, 795-805. Horrobin, D.F., Glen, A.I.M., Glen, E.M.T. and Skinner, F. (1993) Red cell membrane fatty acids in schizophrenic patients with mainly positive or mainly negative symptoms. Schizophr. Res. 9, 221. Houtia, N.E., Maziere, J.C., Maziere, C. and Auclair, M. (1988) Phenothiazines inhibit cholesteryl ester formation in J 774 monocyte-like cells. J. Clin. Chem. Clin. Biochem. 26, 673-678. Kaiya, H., Horrobin, D.F., Manku, M.S. and Morse-Fisher, N. (1991) Essential and other fatty acids in plasma in schizophrenics and normal individuals from Japan. Biol. Psychiatry 30, 3577362. Karmiol, S. and Bettger, W.J. (1988) Accumulation of (nminus;9)-eicosatrienoic and docosatrienoic acids in human fibrobalst phospholipids. Lipids 23, 891-898. Kelkar, H., Wakade, C.G., Reddy, R., Schnur, D., Mukherjee, S. and Mahadik, S.P. (1993) Altered membrane phospholipids in fibroblasts from schizophrenic patients. Biol. Psychiatry 33, 99A. Keshavan, M.S., Pettegrew. J.W., Panchalingam, K., Kaplan, D. and Bozik, E. (1991) 31P NMR spectroscopy detects altered brain metabolism before onset of schizophrenia. Arch. Gen. Psychiatry 48, 1112-I 113. Kuksis, A. (1978) Fatty acid composition of glycerolipids of animal tissues. In: Kuksis, A. (Ed.), Handbook of Lipid Research, Vol. 1, Pergamon, New York, pp. 381-442. Lange, Y., Swaisgood, B.V., Ramos, B.V. and Steck, T.L. (1989) Plasma membrane contains half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts. J. Biol. Chem. 264, 3786-3793. Mahadik, S.P., Hawyer, D.B., Hungund, B.L., Li, Y.S. and Karpiak, SE. (1989) GM1 ganglioside treatment after global ischemia protects changes in membrane fatty acids and properties of (Na+,K+)-ATPase and Mg’+-ATPase. J. Neurosci. Res. 24, 402-412. Mahadik, S.P., Mukherjee, S., Laev, H. et al. (1991) Abnormal growth and morphology of fibroblasts from schizophrenic patients. Psychiatry Res. 37, 309-320. Mahadik, S.P., Mukherjee, S., Wakade, C.G.. Laev, H. and Reddy, R. (1994) Decreased adhesiveness and altered cellular distribution of fibronectin in fibroblasts from schizophrenic patients. Psychiatry Res., in press. Mahadik, S.P., Wakade, C.G., Scheffer, R., Correnti, E., Borison, R.L. and Mukherjee, S. (1993a) Abnormal growth of skin fibroblasts from drug-naive psychotic patients. Biol. Psychiatry 33, 69A. Mahadik, S.P., Hungund, B.L., Gokhale, V.S., Ortiz, A., Makar, T.K. and Karpiak, SE. (1993b) Monosialoganglioside (GMl) restores membrane fatty acid levels in ischemic tissue after cortical focal ischemia in rat. Neurochem. Intern. 23, 163-172. Manku, MS., Horrobin, D.F., Huang, Y.-S. and Morse, N.

(1983) Fatty acids in plasma and red cell membranes in normal humans. Lipids 18, 906-908. Maziere, C., Maziere, J.-C., Mora, L., Auclair, M. and Polonovski, J. (1988) Trifluoperazine increases fatty acid turnover in phospholipids in cultured hyman fibroblasts. Lipids 23, 419-423. Mukherjee, S., Reddy, R. and Schnur, D. (1991) Developmental model of negative syndromes in schizophrenia. In: Greden, J., Tandon, R. (Eds.), Negative Schizophrenic Syndromes. Am. Psych. Press, Washington, DC, pp. 175-185. Mukherjee, S., Scheffer, R., Mahadik, S.P., Correnti, E., Guill, M.A. and Wakade, C.G. (1993) Fibroblast Studies in First Break Psychoses. APA. Neuringer, M., Conner, W.E., Lin, D.S., Barstad, L. and Luck, S. (1986) Biochemical and functional effects of prenatal and postnatal 03 fatty acid deficiency on retina and brain in rhesus monkeys. Proc. Natl. Acad. Sci. USA 83,4021-4025. O’Brien, J.S. and Sampson, E.L. (1965) Lipid composition of the normal human brain: Gray matter, white matter, and myelin. J. Lipid Res. 6, 537-544. Pangerl, A.M., Steudle, A., Jaroni, H.W., Rufer, R. and Gattaz, W.F. (1991) Increased platelet membrane lysophosphatidylcholine in schizophrenia. Biol. Psychiatry 30, 837-840. Pettegrew, J.W., Keshawan, M.S., Panchalingam, K., Strychor, S., Kaplan, D.B., Tretta, M.G. and Allen, M. (1991) Alterations in brain high-energy phosphate and membrane phospholipid metabolism in first-episode, drug-naive schizophrenics. Arch. Gen. Psychiatry 48, 5633568. Rouser, G., Kritchevsky, G. and Simon, G. (1967) Quantitative analysis of brain and spinach leaf lipids employing silicic acid column chromatography and acetone for elution of lipids. Lipids 2, 37-40. Rouser, G., Simon, G. and Kritchevsky, G. (1969) Species variations in phospholpid class distribution of organs. Lipids 4, 599-606. Rotrosen, J. and Wolkin, A. (1987) Phospholipid and prostaglandin hypotheses of schizophrenia. In: Meltzer, H.Y. (Ed.), Psychopharmacoloy: The Third Generation of Progress, Raven, New York, pp. 7599764. Sinclair, A.J. (1975) Incorporation of radioactive polyunsaturated fatty acids into liver and brain of developing rat. Lipids 10, 175-184. Stevens, J.D. (1972) The distribution of phospholipid fractions in the red cell membrane of schizophrenics. Schizophr. Bull. 6, 60-61. Suzuki, K. (1981) Chemistry and metabolism of brainlipids. In: Siegel, G.J., Albers, R.W., Agranoff, B.W., Katzman, R. (Eds.), Basic Neurochemistry, 3rd edn. Little, Brown and Company, Boston, pp. 355-370. Thompson, G.A. (1992) The Regulation of Membrane Lipid Metabolism. 2nd edn., CRC Press, Boca Raton, FL. Van Kammen, D.P., Yao, J.K. and Goetz, K. (1989) Polyunsaturated fatty acids, prostaglandins and schizophrenia. Ann. N.Y. Acad. Sci. 559, 411-424.

S. P. Mahadik et al.lSchizophrenia Research 13 (1994) 239-247 Wainwright, P.E. (1992) Do essential fatty acids play a role in brain and behavioral development? Neurosci. Biobehavioral Revs. 16, 193-205. Wakade, C.G., Ahmed, A., Reddy, R., Schnur, D., Mukherjee, S. and Mahadik, S.P. (1993) Response of fibroblasts from schizophrenic patients to growth factors. Biol. Psychiatry, 33, 99A. Weinberger, D.R. (1987) Implications of normal brain developement for the pathogenesis of schizophrenia. Arch. Gen. Psychiatry, 44, 660-669.

247

Wiesel, F.-A., Blomquist, G., Venizelos, N., Bjerkenstedt, L., Halldin, C., Hagenfeldt 1. and Sjogren, I (1991) Altered transport of tyrosine across the blood-brain barrier in patients with schizophrenia. In: Racagni, G., Brunello, N., Fukuda, T. (Eds.), Biological Psychiatry, Vol. 2, Excerpta Medica, Amsterdam, pp. 337-340. Williamson, P., Drost, D., Stanley, J. et al., (1991) Localized phosphorus 31 nuclear magnetic resonance spectroscopy in chronic schizophrenic patients and normal controls. Archieves of Gen. Psychiatry 48, 578 [Letter].