Effect of Lead on Lipid Peroxidation, PhospholipidsComposition, and Methylation in Erythrocyte of Human

Shafiq-ur-Rehman

Received: 10 April 2013 /Accepted: 25 June 2013 /Published online: 12 July 2013# Springer Science+Business Media New York 2013

Abstract Lead (Pb) is one of the most abundant heavymetals on earth considered as number one environmentalpersistent toxin and health hazard affecting millions of peo-ple in all age groups. After entering bloodstream, 99 % of Pbis accumulated in erythrocytes and causes poisoning. ToxicPb effects on erythrocytes membrane’s composition of phos-phatidyl serine (PS), phosphatidyl ethanolamine (PE), phos-phatidyl choline (PC), and sphingomyelin (SM), and phospho-lipids transmethylation were determined. Lipid peroxidation inPb-exposed erythrocytes was evaluated as malondialdehyde(MDA) formation in presence of Fe and vitamin E to under-stand severity of Pb toxicity and its mitigation. Pb (0.5–5.0 μM) degraded PS (12 to 31 %, P<0.05–0.001) and elevat-ed SM (19–51 %, P<0.05–0.001). Composition of PC and PEwere diminished (22%) and elevated (29%), respectively, withhigher Pb exposure (5.0 μM, P<0.001). Pb toxicity suppressed(P<0.001) transmethylation of phospholipids in membranes(34, 41, and 50 %, respectively, with 0.5, 2.5, and 5.0 μM).Pb-induced dose-related MDA production (P<0.05–0.001) inerythrocytes was obtained, which was accentuated in presenceof Fe (P<0.05–0.001). The vitamin Emitigated (P<0.05–0.01)the severity of Pb-induced lipid peroxidation. The ratio PS/SMshowed maximum change of −27 (P<0.01), −30 (P<0.01),and −54 % (P<0.001), respectively at 0.5, 2.5, and 5.0 μM Pbexposures. Ratios PC/SM and PS/PE were at the second,whereas PE/PS at the third order. The study suggests that themechanisms underlying distortion of compositional phospho-lipids, inhibition of transmethylation, and exasperated phos-pholipid peroxidative damage are the active phenomena of Pbtoxicity in erythrocytes.

Keywords Alpha-tocopherol . Erythrocyte membrane .

Lead toxicity . Phospholipids composition .

Transmethylation . Oxidative stress

Introduction

Lead (Pb) is one of the most abundant heavy trace metals onearth considered as number one environmental persistent tox-in and health hazard affecting millions of people in all agegroups. A recent estimate by the World Health Organisationhas revealed that 120 million people are over-exposed to Pb,approximately three times the number infected by HIV/AIDS,and 99 % of the most severely affected are in the developingworld [1]. Once it enters the blood stream, 99 % of Pb is takenup by the erythrocytes and the remaining 1 % of the Pb is leftwith plasma [2]. This probably makes the erythrocytes highlyvulnerable to Pb poisoning. Result of a recent study indicatedthat Pb ions adhered to the external and internal surfaces oferythrocyte membrane [3]. This shows that Pb can influencemolecules present in both layers of the membrane. Attributingto high affinity of Pb for sulfhydryl-containing molecules [4], Pbhas been shown to be interacted with heme, delta-aminolevulinicacid dehydratase, (Na+-K+)-ATPase, and adenylatecyclase en-zymes, and the processes of ions transport in erythrocytes [5–8].

Studies have shown that Pb exposure has decreased [9] ornot affected [10] phospholipid level in erythrocyte cell mem-brane. We have previously reported that Pb has degradedphospholipid in the regions of brain those facilitated Pb accu-mulation [11]. There is scarce report on ill effects of Pb inerythrocyte cell membranes in respect to composition of thephospholipids that carry functional and metabolic role in the cellmembranes. Phospholipids are asymmetrically distributed in themembrane bilayer of erythrocyte, with amino-containing phos-phatidyl serine (PS) and phosphatidyl ethanolamine (PE) on theinner cytoplasmic and choline-containing phosphatidyl choline(PC) and sphingomyelin (SM) on the exterior extracellular

Shafiq-ur-Rehman (*)Division of Environmental Sciences, Section of EnvironmentalHealth and Toxicology, Sher-e-Kashmir University of AgriculturalSciences and Technology of Kashmir, Srinagar 191121, Jammu andKashmir, Indiae-mail: shafiqurrehman11@yahoo.com

Biol Trace Elem Res (2013) 154:433–439DOI 10.1007/s12011-013-9745-1

surfaces. These phospholipids structurally provide a molecularstrength to the membrane stability and functional proteins andenzymes [12, 13].

Phospholipids could display a dynamic role during theirsynthesis, namely, the PC synthesis from PE through a highlyactive process of transmethylation. The trans-methylation pro-cess of PE synthesis to PC maintains asymmetric distributionof the phospholipids throughout the membrane bilayer [14]. Inthis process, enzyme methyltransferases I and II play a signif-icant role in trans-location (or flip-flop) of phospholipid acrossthe membrane bilayer [15, 16]. The process of trans-methylation pathway accomplishes with the addition of threemethyl groups from S-adenosyl-L-methionine (SAM; themethyl donor) to the amino moiety of PE molecule. Themethyltransferases-I (MT-I) having high affinity for SAMmethylates cytoplasmic PE to phosphatidyl-N-monomethylethanolamine (PNE). The methyltransferases-II (MT-II) trans-fers methyl group from SAM to PNE to phosphatidyl-N1-N2-dimethyl ethanolamine (PNNE) and finally to PC at the outermembrane [14]. Studies have also shown that phospholipidscould display a dynamic role during their synthesis (e.g. PCfrom PE) through the highly active process of trans-methylation and could, therefore, elucidate important mecha-nism for biological signal transduction across the membrane[17, 18], instigating a number of biological events. In schizo-phrenic illness, for example, decreased PC composition wasrelated to decreased phospholipids methylation in erythrocytemembranes [19]. However, the mechanism of compositionaltransformation of phospholipids classes and their transloca-tion through bilayer membranes of erythrocytes cell via theprocess of methylation is not yet elucidated in the event of Pbtoxicity. The Pb-induced degradation of regional phospholipidand augmentation of lipid peroxidation has been reported in thebrain [11, 20, 21], whereas these events were not seen inerythrocytes membranes [10]. The degradation of structuralphospholipids classes and their relation with oxidative stress inthe erythrocytes membranes are still not well established in Pbexposure. However, lipid peroxidation, which is a highly dam-aging peroxidative process of phospholipids, could take placevia free radical-mediated oxidative injury to biological systemsin certain patho-toxicological states [22, 23]. Nevertheless, theerythrocyte membranes could particularly be highly sensitive tooxidative damage from lipid peroxidation because of their highcontents of polyunsaturated fatty acids and heme iron concen-trations as well as continuous exposure to high concentrations ofoxygen [24, 25]. It was therefore assumed that the vulnerableenvironment of erythrocytes could easily facilitate toxic aptitudeof Pb to provoke the generation of reactive oxygen species inorder to instigate cycles of lipid peroxidation reactions and todamage composition of membrane phospholipids and theirmethylation leading to oxidative injuries, and therefore, thiscould provide the important mechanism of Pb toxicosis. Thepresent study appraises these aspects.

Materials and Methods

Materials

S-adenosyl-L-[methyl-3H] methionine (60 Ci/mmol) waspurchased from Amersham Buckler GmbH & Co KG, Ger-many. All other chemicals and reagents were of analytical orHPLC grade.

Separation of Erythrocytes and Pb Exposure

Blood samples in tubes with heparin were taken (with in-formed consent) from healthy male (30–35 years) donors atthe hospital facility. Erythrocytes were separated by washingwith phosphate buffer (5 mM sodium phosphate and150 mM NaCl (pH 8.0)) as described in detail earlier [26].Washed-packed erythrocytes were used in all analyses. Forexperimental manipulations, the packed cells were exposedto Pb (0.5, 2.5, or 5 μM corresponding to 10∼50 μg/dl oftoxicological significance; N=4 to 6 or otherwise mentioned)incubating at 37 °C for 30 min or otherwise indicated. Theseexposure doses of Pb are closed to Fiorini et al. [27] and Shinet al. [28].

Preparation of Human Erythrocyte Membranesand Extraction of Phospholipids

The packed cells were haemolysed by thorough mixing in 40volumes of the phosphate buffer. The white creamy intactghost membranes were obtained after three to four identicalwashes as described by Steck and Kant [29]. Phospholipidswere extracted from erythrocyte ghost membranes in chlo-roform, methanol, and water system as described in detail byShafiq-ur-Rehman [26].

High-Performance Liquid Chromatographyof Phospholipids

The phospholipids from the extracts of erythrocyte ghostmembranes were separated and determined by the isocratichigh-performance liquid chromatography (HPLC)-hyphen-ated UV method of Shafiq-ur-Rehman [26]. The mobilephase of acetonitrile–methanol–85 % phosphoric acid(100:10:1.8, v/v) composition was delivered to the silica-based (5 μm spherical particles of 80 Å pore) column at aflow-rate of 1.5 ml/min. The detector wavelength was set at203 nm.

Methylation of Phospholipids in Erythrocyte Membrane

The packed erythrocyte ghost membranes (300 μl) weresuspended in 700 μl of 10 mM MgCl2-50 mM tris-glycylglycinebuffer, pH 8.0, with 4 μCi S-adenosyl-L-[methyl-3H]

434 Shafiq-ur-Rehman

methionine and pre-incubated overnight at 4 °C. Total meth-ylation was measured as described by Hirata and Axelrod [14].

Lipid Peroxidation Assay

Assay of malondialdehyde (MDA) formation was performedby 2-thiobarbituric acid (TBA) reaction as described byShafiq-ur-Rehman [11]. The packed erythrocytehomogenateshaving Pb (0.5, 2.5, or 5 μM), with or without 50 μg Fe (II) or100 μg vitamin E (vit E; in separate experiments) were aero-bically incubated for 2 h at 37 °C. The TBA reaction specieswas recorded at 535 nm. The results were expressed as n molof MDA formed per 30 min; the molecular extension coeffi-cient of MDA expressed as E535=1.56×10

5 [30].

Statistical Analysis

Statistical analysis was performed using Statistical Packagefor Social Sciences (SPSS, 11.5 version), SPSS Inc, Chica-go, IL. Data were subjected to paired-samples t test andANOVA. Relationship between Pb exposure and MDA for-mation levels were tested by linear regression analysis. Re-sults were demonstrated in terms of mean+SE (standarderror) of mean. Differences between tests and correspondingcontrols were significantly compared if the p values wereless than 0.05. Phospholipids compositions of erythrocytemembrane and lipid peroxidation in normal and different Pbexposure levels were accessed in the box–whisker plots forcomprehensible presentation of the findings and the statisti-cal characteristics depicting different categories of data. Thebox plot is a convenient way of graphically depicting groupsof numerical data with more detailed information consistingof the minimum, first quartile (Q1), median, third quartile(Q3), and maximum.

Results

Effects of Pb on Phospholipids Composition of ErythrocyteMembrane

In the present study, the composition of phospholipids in thenormal human erythrocyte membranes was evaluated as PS26.53 %, PE 26.81 %, PC 32.09 %, and SM 14.47 % by theHPLC-hyphenated UV technique (Fig. 1a–d). All the threeconcentrations of Pb (0.5, 2.5, or 5 μM) significantly de-creased the PS (12, 17, and 31 %, P<0.05, P<0.001, andP<0.001, respectively) (Fig. 1a).The high concentration ofPb (5 μM), however, significantly diminished the PC (22 %,P<0.001) composition of the erythrocyte membranes (Fig. 1-c). The SM composition of the erythrocyte membranes wassignificantly increased by 19 (P<0.01), 19 (P<0.01), and51 % (P<0.001), respectively, with 0.5, 2.5, and 5 μM Pb

exposure (Fig. 1d). The 5 μM Pb exposure, however, signif-icantly enhanced the PE (29 %, P<0.001) composition of theerythrocyte membranes (Fig. 1b).

Anomaly of Phospholipids Ratio in Erythrocyte Membraneby Pb

The ratios of the compositional and structural phospholipidsof the erythrocyte membrane represented sharp assessment ofdifferent levels of Pb exposure as shown in the clusteredcolumns (Fig. 2). The ratios of PS/SM, PC/SM, and PS/PEshowed significant reduction with all the exposure doses of Pb(0.5, 2.5, and 5.0 μM), whereas PC/PE ratio was significantlyreduced with maximum exposure dose as compared withcorresponding controls. The ratio PS/SM showed significantlymaximum change of −27 (P<0.01), −30 (P<0.01), and−54 % (P<0.001), respectively, at 0.5, 2.5, and 5.0 μM Pbexposures. Other ratios such as PC/SM and PS/PE were at thesecond order of changes, whereas PE/PS ratio ranked third.However, all the ratios exhibited maximum change at maxi-mum dose of Pb exposure.

Suppression of Phospholipids Methylation in ErythrocyteMembrane by Pb

Methylation process of the phospholipids synthesis in theerythrocyte membranes was significantly (P<0.01) inhibitedby Pb (0.5, 2.5, or 5 μM) in a dose-dependent manner.Detailed observations showed that Pb depressed the phos-pholipids methylation in erythrocyte ghost membranes by34.10 % (P<0.001) at 0.5 μM, 41.35 % (P<0.001) at2.5 μM, and 50.20 % (P<0.001) at 5 μM (Fig. 3).

Pb-Induced Formation of Lipid Peroxidation in ErythrocyteCell

The MDA values were significantly enhanced in erythrocytemembranes by increasing concentrations of Pb. The relation-ships between the impact of different concentrations of Pbexposure (0.5, 2.5, and 5.0 μM) and the formation of MDA inthe erythrocytes showed a linear pattern (presented as line inFig. 4) of regression test as, MDA=1.296x+0.745 Pb with amultiple R2=0.9934 and significant level less of than 0.0001.Paired sample statistics reveal that the impact of Pb exposureon the erythrocyte membrane was significant with concentra-tions of 0.5 (P<0.05), 2.5 (P<0.01), and 5 μM (P<0.001)when compared with controls, showing a dose-dependentincrease of the MDA formation of 46.51, 118.60, and176.74 %, respectively. The MDA formation was enhancedin erythrocyte membranes under pro-oxidant system (Fe II) by25.57 % (P<0.05) when compared with controls. Pb exagger-ated the Fe-induced MDA formation by 64.81 (P<0.01),112.96 (P<0.001), and 184.44 % (P<0.001), respectively,

Lead and Human Erythrocyte Membranes 435

with treatments of 0.5, 2.5, and 5 μM Pb concentrations.Under anti-oxidant system, however, Pb-mediated formationsof MDA in erythrocytes were significantly alleviated(P<0.5 at 0.5 μM and P<0.01 at 2.5 and 5 μM) by thescavenger vit E (Fig. 4).

Discussion

Erythrocyte membrane is asymmetrically constructed in abilayer manner with amino-phospholipids (PS and PE) onthe inner cytoplasmic and choline-containing phospholipids

A

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Fig. 1 Effects of differentconcentrations of Pb (0.5, 2.5, or5.0 μM) on erythrocytemembrane composition ofphospholipids (a PS, b PE, c PC,and d SM) are shown. Thesignificant changes wereassessed at P less than 0.05 (onestar), 0.01 (two stars), and 0.001(three stars) levels. Per centchanges of individualphospholipids (mean+SE) aregiven for each concentration ofPb exposure comparing with thecontrol group (Pb, 0), N=6

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Fig. 2 The ratios ofcompositional phospholipids inhuman erythrocyte membranesbefore (normal) and afterexposure of differentconcentrations of Pb (0.5, 2.5, or5.0 μM) are represented (N=6).The data are values representedas mean+SE with 1* for P<0.05,2* for P<0.01, and 3* forP<0.001, at 5 % level ofsignificance

436 Shafiq-ur-Rehman

(PC and SM) on the exterior extracellular surfaces. Thetypical arrangement of these phospholipids remains stablethroughout the membrane that maintains mixed physico-chemical expressions of the cell. Albeit, the fatty acid con-stituents of the phospholipids, in general, are labile and canbe modified by changing dietary pattern [31], on the otherhand, the base groups (amino and choline) may be altered indiseased states [19]. In Pb toxicity, the degradation of phos-pholipid has been reported to be evident in those regions ofthe brain which absorbed Pb [11]. Extremely high percentage(approximately 99 %) of Pb in the blood is bound witherythrocytes. Pb has been shown to adhere on both surfacesof the human erythrocyte cell membrane incubated with highdose of Pb [3]. The present investigation demonstrates thatwith the moderate doses (i.e. 0.5–5 μM) Pb has had easyaccess to the outer as well as the inner membrane surfaces ofthe erythrocyte cell, as Pb remarkably influenced the com-positions of both the outer and inner membrane phospho-lipids of human erythrocytes. Detailed observations havefurther revealed that Pb degraded the amino-containingphospholipid PS at the interior cytoplasmic layer and thecholine-containing phospholipid PC at the exterior extracel-lular surface of the human erythrocyte membranes. Thisattributes that Pb binding with the PS at the inner membranenot only degraded the PS composition but also the PC at theouter membrane surface of the erythrocyte. This may denote

essential mechanisms of antagonistic sway of the Pb on thePS and PC molecules and biological functions associatedwith these phospholipids.

The PE composition of the erythrocyte membranes wasfound elevated whereas the PC levels were declined by Pbexposure. The transmethylation activity that leads transforma-tion of PE to PC was inhibited by Pb in the present study. Thisshows that the PE was not utilised or barred from taking part inthe process of methylation for PC synthesis. Since the phos-pholipids are successively methylated, they are translocatedfrom the inside to the outside of the membrane [17]; this wasnot happening in the present circumstances of Pb toxicity. Thisshows that Pb ability of interfering or binding with the thiolcontaining molecule might have seized the methyl transferfacility of the SAM to PE, PNE, and to PNNE molecules.Similar inhibiting capability of Pb cannot be ruled out for theenzymes MT-I and MT-II, as well. The inhibition of phospho-lipids transmethylation in erythrocyte membranes was increas-ingly prominent with the increasing concentration of Pb expo-sure. The observed inhibition of phospholipids transme-thylation by Pb seemed to have seized the phospholipids syn-thesis in the erythrocyte membranes. The correlation betweendecreased composition of PC and the decreased transme-thylation of phospholipids [17] could be established as a mech-anism of Pb toxicity in erythrocyte membranes as evidentlyshown in the present study. The suppression of phospholipidstransmethylation appeared to be of particular appealing sincethis process could in fact affect a wide range of membranesfunctions. Likewise, phospholipids methylation has been

-60

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Fig. 3 Inhibitory effects of Pb on trans-membrane methylation of phos-pholipids in human erythrocyte membranes. The inhibitory assessment ofPb was carried out with three different concentrations of Pb (0.5, 2.5, or5.0 μM) exposure (values are expressed as mean±SE; Star P<0.001,significant values N=4)

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Fig. 4 Effects of 0.5, 2.5, or 5.0μM treatments of Pb to erythrocyte showa strong dose response increment of lipid peroxidation (mean+SE) asshown in linear regression pattern (solid line). Observations under pro-and anti-oxidant regime, carrying different treatments of Pb (0.5, 2.5, or5.0 μM) alone or in the presence of Fe II (50 μg), or vit E (100 μg) witherythrocyte membranes, reveal more aggression of lipid peroxidativeinjury or its mitigation to the affected membranes, respectively. Signifi-cance was determined by paired samples test (*P<0.05; **P<0.01;***P<0.001 compared with groups Pb vs. Fe II or Pb vs. vit E, N=4)and ANOVA (P<0.001)

Lead and Human Erythrocyte Membranes 437

implicated in regulating the response of adenylatecyclase toactivate β-adrenergic receptor [32]. Formation of cAMP hasalso been activated by phospholipids methylation. Studies withβ-adrenergic antagonist system (propranalol) have shown theinhibition of phospholipids methylation and cAMP formation[14]. On the other hand, Pb has been implicated in reducing thelevels of cAMP and inhibiting the β-adrenergic receptors [33].It appeared likely that the inhibition of phospholipids methyl-ation by Pb could affect the β-adrenergic receptor function inmembranes. The interaction of Pb with membranes couldreveal a novel toxicological significance of Pb in the phospho-lipids transmethylation activity (i.e. flip flop or migration ofphospholipids across the membrane surfaces) and associatedphysiological functions. Therefore, Pb could express impres-sion on phospholipids transmethylation and associated eventsas well, for example, calcium-evoked release of histamine [16].

In healthy erythrocyte cells, transmethylation activity of PCsynthesis maintains PC and SM composition at the extracellu-lar side of the membrane. However, the transformation intransmethylation activity due to Pb exposure (i.e. toxicity) hasdisturbed the normal composition of phospholipids of themembranes as evidenced in this study. Moreover, the SMcomposition of the membrane was found increased in Pbintoxication. It has been shown that the deficiency of PC inthe membranes was largely compensated by the elevated levelsof SM composition in order to maintain the lipid-bound choline[19]. Therefore, the characteristic of SM in the functional andstructural stabilisation of erythrocytes membranes describes animportant role in Pb toxicity. The compositional anomaliesof PS, PE, PC, and SM in the erythrocyte membraneshave consequently affected the phospholipids ratios. ThePS/SM, PC/SM. and PS/PE ratios showed diminished valuesin all the exposure doses of Pb. However, PC/SM ratio wasfound reduced only at the maximum exposure dose of Pb.Hitzemann and co-workers [19] have shown altered ratios ofthe phospholipids in schizophrenia. Pb exposure has beenfound to develop schizophrenia-like complex stereo-typedbehaviours in rats [34]. This apparently reveals that the ob-served changes of phospholipids ratios in the erythrocytemembranes could implicate their importance in the mecha-nism of Pb toxicosis.

One of the major mechanisms underlying heavy metaltoxicity has been attributed to oxidative stress. Lipid perox-idation is one of the most important organic expressions ofthe oxidative stress caused by the reactivity of free radicalswith molecules of biological systems that come under attackof metal [35]. We have explored earlier the increased forma-tion of MDA as an index of lipid peroxidation in Pb-inducedoxidative damage to the brain [20, 21]. Moreover, the deg-radation of phospholipid and exceeded generation of MDAfollowing the hyper-activated process of lipid peroxidationin the brain regions have provided a convenient model tostudy the mechanisms of Pb-induced oxidative damage to

biological cell membranes [11]. The membrane phospho-lipids, containing high levels of polyunsaturated fatty acids,are predominantly susceptible to peroxidation damages. Inview of these events, the erythrocytes were employed in thepresent study because of the simplicity, availability, and easeof isolation make erythrocyte membrane as an excellentmodel for membrane studies [36]. Moreover, erythrocytesand erythrocyte membrane are more vulnerable to lipid per-oxidation because of constant exposure to high oxygen ten-sion and riches in polyunsaturated fatty acids [24, 25]. Fur-thermore, altered lipids composition of cellular membranesmay alter membrane integrity, permeability, and function. Inthe present study, the compositional phospholipids of eryth-rocyte membranes were found highly affected by Pb expo-sure. Among these phospholipids in erythrocytes membranes,the PS and PC were significantly degraded (∼31 and 22 %,respectively, P<0.001, see Fig. 1a, c) in Pb toxicity. Furtherinvestigation indicated concurrent production of MDA in adose–response manner of increasing Pb exposure, following alinear regression patternwithR2=0.9934 and significant level of<0.0001. Specifically, when compared with the control, signif-icant increasing values ofMDA formation have been shown (by47 % P<0.05, 119 % P<0.01, and 177 % P<0.001) withincreasing exposure of Pb (0.5, 2.5, and 5 μM, respectively).The tendency of Pb toxicity causing lipid peroxidation in eryth-rocytes was further validated with primary oxidant system (FeII) and found that Pb significantly exaggerated the Fe II-inducedlipid peroxidation in erythrocytes, which validated our earlierclaim that Pb toxicity induces lipid peroxidative damage [11, 20,21]. Furthermore, the anti-oxidant system scavenger vit E sig-nificantly mitigated the severity of peroxidative stress of Pbtoxicity in erythrocytes. The behaviour of lipid peroxidation inerythrocyte membranes in the presence of pro- and anti-oxidantsystems further validates our argument for the mechanism un-derlying formation of the end product of lipid peroxidationMDA as an index of Pb injury to biological membranes.

Acknowledgements The author is highly grateful to Professor Dr. M.Ackenheil for continuous encouragement and help. This study is dedi-cated to his affectionate memory. Thanks are also due to Ms. ShaheenRehman for help in analysis and preparation of the manuscript. The workwas performed at the Department of Neurochemistry, Institute of Psychi-atry, Ludwig Maxmilian University of Munich, Germany.

Conflict of Interest The author reports no conflict of interest.

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