EFFECT OF FREE FATTY ACIDS ON ERYTHROCYTE MORPHOLOGY AND MEMBRANE FLUIDITY. tlya B. Zavodnik*, Andrzej Zahorowski, Andrzej Niekurzak, Maria Bryszewska

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1 Vol. 42, No. 1, June 1997 Pages EFFECT OF FREE FATTY ACIDS ON ERYTHROCYTE MORPHOLOGY AND MEMBRANE FLUIDITY tlya B. Zavodnk*, Andrzej Zahorowsk, Andrzej Nekurzak, Mara Bryszewska Insttute of Bophyscs, Unversty of Lodz, 12/16 Banacha St., Lodz, Poland, *Insttute of Bochemstry, Academy of Scences of Belarus, BLK - 50, , Grodno, Belarus Receved February 10, 1997 Receved after revson February Summary: Changes n red blood cell shape and membrane propertes n response to the nteracton wth free fatty acds and ther dervatves were studed by lght scatterng at small and large angles, lght mcroscopy and fluorescence ansotropy. The nfluence of these agents depended on the end groups and ncreased wth ncreasng chan length. The fatty acds exerted a bphasc effect on the cell sze, shape and surface propertes, and nduced erythrocyte aggregaton. After transent sze alteraton wth a reducton n dameter, caused by low free fatty acd concentratons (up to 5-10 gm n the case of palmtc acd), fatty acds ncreased the erythrocyte dameter at hgher concentratons (20-60 gm n the case of palmtc acd). The alphatc aldehydes and methyl esters of fatty acds sgnfcantly decreased the cell dameter at the concentratons used. Changes n erythrocyte shape and sze were accompaned by changes n membrane mcrovscosty. Palmtc acd decreased the rotatonal dffuson of the fluorescence probe ncorporated nto the membrane whereas methyl ester of palmtc acd and laurc aldehyde ncreased probe moblty. Also the erythrocyte modfcaton by malondaldehyde nfluenced cell morphology and hghly decreased membrane fludty. Key Words: red blood cells; morphology; membrane; free fatty acds. Introducton Interactons of the free fatty acds (FFA), ther dervatves and varous other amphphlc molecules wth red blood cells have been studed n many laboratores. Snce the early papers of Seeman et al. [1] t has been known that agents wth an amphphlc character nteract wth the erythrocyte membrane n a bphasc way. They protect erythrocytes aganst hypotonc haemolyss and nduce lyss of the cells n sotonc medum [1-4]. At the same tme, the mechansm (or mechansms) underlyng the haemolytc and anthaemolytc effects of FFA and ther dervatves as well as the common mechansms of cell changes due to the nteractons wth FFA are not fully understood. For correspondence: Dr. Mara Bryszewska, Unversty Bophyscs, 12/16 Banacha St., Lodz, Poland tel , e-mal <marbrys@bol.un.lodz.pl> 123 of Lodz, Department of Medcal /97/ I I $05.00/0 (-;ol~yl'ghl ~2~ 1997 hy Academc Pvss Australa All rghts <!/repod.<'fojz n any fnm resel l'ed

2 A membrane/aqueous phase partton s the mechansm whereby the amphphle monomers ntercalate nto the membrane [2]. At anthaemolytc concentratons amphphles ncrease potassum permeablty of the erythrocyte membrane [2] and nduce a release of exo- and endovescles as a result of detachment of the cytoskeleton network from the membrane [3-5]. Palmtc, olec and lnolec acds can be ncorporated nto the membrane at concentratons up to 1000 nmol of FFA / ml packed cells wthout causng any sgnfcant haemolyss and wthout any sgnfcant changes n the actvty of the sodum pump of human erythrocyte membranes [6]. The marked shape changes of the erythrocytes n the presence of amphphles precede vcsculaton [3,5]. Loadng of erythrocytes wth phosphatdylcholne, lysophosphatdylcholne or stearc acd nduces an apprecable crenaton of the red blood cells and causes nhbton of glucose transport across the membrane. Ths effect of the loaded lpds on glucose transport may not have a drect connecton wth the change of the cell shape [7]. At the same tme, the anthaemolytc potency of the amphphles s not related to the type and extent of shape changes nduced by these agents [3]. The am of ths work was to study the changes of the red blood cell shape, sze and refractve propertes n the presence of FFA and ther dervatves, usng the methods of flow cytometry and fluorescence ansotropy. The effect of long chan effectors was compared wth the effect of malondaldehyde (MDA), the hghly toxc and stable end - product of lpd peroxdaton, whch nduces nonenzymc modfcaton of the protens and membrane consttuents [8]. Materals and methods Chemcals of analytcal grade were obtaned from POCH (Glwce, Poland), Molecular Probes, Eugene, Oregon, USA (1-[4-trmethylammonumphenyl]-6-phenylhexa-l,3,5-trene, TMA- DPH), Reachm, Russa (fatty acds and ther methyl esters ), Ferak, Berln (laurc aldehyde). Malondaldehyde was obtaned [8] by acd hydrolyss of 1,1,3,3-tetramethoxypropane (Aldrch, Germany). Blood samples from healthy ndvduals were obtaned from the Central Blood Bank of Lodz. Erythrocytes were washed three tmes wth sodum phosphate-buffered salne (PBS: 0.15 M NaC1, 1.9 mm NaH2PQ, 8.1 mm Na2HPQ, ph 7.4) and suspended n PBS. Fatty acds and ther dervatves were added to red blood cell suspensons as concentrated ethanolc solutons. The fnal ethanol concentraton dd not exceed 1%. The erythrocytes were modfed by MDA by ncubaton of cell suspensons (haematocrt of 1% ) n PBS wth varous concentratons of MDA at 37 ~ for 1 hour. The suspensons were then dluted wth cold PBS up to haematocrt of 0.05%. Flow cytometry. The analytcal technque of flow cytometry has become essental for studes of cells by means of lght scatterng and fluorescence spectrophotometry. Detecton of scattered lght yelds nformaton about cell sze and shape. When a cell passes through the exctaton beam n a flow cytometer, t scatters lght n all drectons. The lght s typcally detected 124

3 Vol. 42, No. ]., ] 997 BIOCHE.MISTRYond MOLECULAR BIOLOGY IN'I'ERNA1]ONAL n the forward drecton and at rght angles to the ncdent beam. The scattered rradance near the forward drecton depends on cell sze and s approxmately ndependent of cell refractve ndex and shape [9]. The scattered rradance at larger angles s more senstve to nternal structural dfferences, refractve ndex dfferences and cell shape. In order to evaluate the sze and shape changes of a red blood cell as affected by free fatty acds and ther dervatves, we used a Scatron Argus (Norway) arc lamp-based compact flow cytometer wth smultaneous separate detecton of low angle (LS1) and rght angle (LS2) lght scatterng. Erythrocyte suspensons at a haematocrt of 0.05% were ncubated wth fatty acds or ther dervatves for 15 mn and lght scatterng dagrams of samples were regstered. Membrane fludty. The suspensons of erythrocytes (haematocrt of 0.05%) were labelled wth the fluorescent probe TMA-DPH by ncubatng at room temperature for 5 mn. The fnal concentraton of the fluorescent label was 1 ~tm. The steady-state fluorescence ansotropy of the label was determned n a Perkn-Elmer LS-5B spectrofluormeter. Polarzers select the vertcal and horzontal components of both the exctaton (356 nm) and the emsson (428 nm) beams, The ansotropy was calculated accordng to: r = (Ivy - Ivu x G)/(Ivv + Ivnx G) where Ivv and Ivn are the components of emtted lght ntensty, parallel and perpendcular respectvely, wth reference to the drecton of polarzaton of the exctaton lght, and G s the correcton factor (G = I.v/Irm ) used to correct for unequal transmsson n the optcs. Lght mcroscopy. Erythrocyte suspensons n PBS (10% haematocrt) at tme zero and after 15 ran of ncubaton wth FFA were fxed and dyed on glass surfaces by the method of May-Grunwald and Gemsa [11]. Then cell samples were vewed under Olympus (Japan) phase contrast mcroscope usng a magnfcaton of 500x. Statstcs. The results are expressed as mean + SD; statstcal sgnfcance of the dfferences was evaluated usng Student's t-test. Results Flow eytometry. Typcal scatter hstograms for a control sample are shown n Fgure 1. LS1 s the dstrbuton of lght scattered- n the forward drecton; LS2 s the dstrbuton at a rght angle; and LS 1/LS2 s a dual parameter contour plot. LS 1 depends on cell sze, whereas LS2 s senstve to cell shape and nternal structural dfferences. For each hstogram, the data wthn the regon defned by the cursors were contnously scanned for computaton of the number of events regstered and percentage relatve to total (w/t). Peak (P) channel poston was also dsplayed. Cursors were set n fxed postons n scatter hstograms for a control (untreated erythrocytes) sample to dentfy the possble appearance of varous subpopulatons of red blood cells as nfluenced by FFA and ther dervatves. Fgs. 2, 3 and 4 summarze the results obtaned. The bphasc effect of FFA on the erythrocyte shape and morphologcal propertes can be seen. At low concentratons ofpalmtc acd (up to 5-10 ~M) the poston of the peak of the LS1 and LS2 hstograms moves to the left relatve to the control cells (more so n the LS1 hstogram) (Fg. 2). At hgher palmtate concentratons the peak moves to the rght. The percentage of cels 125

4 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL -I E I ' 'LS1 -- Q c" ID r- e c- d LS1 I -I 'tlj LS1 g 2O 76 channe~ 2O 76 channel channel b e h G G 0 c- O.o E c o \ 87,'s2- -' LS2 c- - channe[ o 2 -fl J o c 0 t- N o 87 channel channel c [ LSI Ls1 f 0 ks LS2 LS2 Fg. 1. Typcal scatterng hstograms from control erythrocytes (a-c), erythrocytes n the presence of 50 txm palmtc acd (d-f) and of 200 I_tM palmtc acd (g-). a, d, g - Low-angle lght scatterng (LS1); b,, h - rght-angle lght scatterng (LS2); c, f, - LS1/LS2 dual parameter contour plots (planar vew). relatve to total (w/t) between the cursors (the percentage of cells wthn fxed boundares) decreases dramatcally from 98.5% to 28% for 60 ~tm of palmtc acd (Fg. 3). The effect of FFA on the w/t parameter s hgher for rght angle lght scatterng (LS2) than for forward scatter (LS 1). At hgh concentratons of FFA (200 ~tm for palmtc acd) cell haemolyss and ghost formaton can be observed (Fg. 1). The effect of FFA ncreases concurrently wth the ncrease of the length of effector acyl chan. Caprylc acd has an effect on lght scatterng parameters of red blood cells only at very hgh concentratons (400 ~tm, results not shown). 126

5 O c-2 -a--3 2O 10 L [ I I t I [ I C effector, I~M = 40 ~ 30 ~ 20 [-'~- 11!-o-21 --*--3 -:x, I [ I I [ [ I t Ceffector, ~,~/~ Fg. 2. Poston of the peak (P) of (a) low-angle lght-scatterng hstogram (LS l) and (b) rght-angle lght- scatterng hstogram (LS2) n the presence of caprylc acd (1), palmtc acd (2), methyl ester of palmtc acd (3) and laure aldehyde (4). 127

6 o-- 1, --o-2!. m 40,! I 1 I L I 1 [ I Cefrector, gm Fg. 3. Percentage of unchanged cells relatve to the total (w/t), measured by rght-angle (LS1) lght scatterng n the presence of caprylc acd (1), palmtc acd (2), methyl ester of palmttc acd (3), and laure aldehyde (4). The effects of alphatc aldehydes and methyl esters of FFA are smlar but they dffer from that of the correspondng FFA. The peak channel poston of LS1 and LS2 lght scatterng hstograms moves to the left for most aldehyde or ester concentratons (Fg. 2). At laurc aldehyde concentratons exceedng 30 ~M fast haemolyss was observed. At the same tme, n the presence of methyl esters two peaks n the scatterng hstograms are observed (data not shown). The second peak, whch appears as effector concentraton ncreases, s shfted to the rght n comparson wth the control one. MDA, whch covalently modfes red blood cell membranes [8], moves the peak poston to the left at low concentratons (up to 0.4 mm), whle hgh concentratons of ths compound shft ths parameter to the rght (Fg. 4). The percentage of cells relatve to total between the cursors remans unchanged for both scatter hstograms after erythrocyte modfcaton by MDA. The postons of the peak and medan changed n lne and the curves of hstograms were symmetrcal for control cells and for cells after modfcaton by FFA (Fg. 1), ther dervatves or MDA. 128

7 ,345 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL ~30 o t/ 20 0L/ l 0 ~ 0,32 0 0,5 1 1,5 2 CMDA, mm Fg. 4. Poston of the peak (P) of the low-angle lght-scatterng hstogram (1), rght-angle lghtscatterng hstogram (2) and ansotropy of the fluorescence of TMA-DPH ncorporated nto membranes (3) for erythrocytes modfed by MDA. Membrane fludty. The nfluence of effectors on red blood cell morphology was compared wth that on erythrocyte membrane fludty measured by fluorescence ansotropy of TMA-DPH ncorporated nto the membranes. It s shown n Fg. 5 that palmtc acd ncreased the fluorescence ansotropy of TMA-DPH, whereas the methyl ester of ths acd and laurc aldehyde decreased t. The change of erythrocyte membrane fludty correlated wth the change of erythrocyte lght scatterng parameters. Modfcaton of erythrocyte membranes by MDA ncreased the value of the TMA-DPH fluorescence ansotropy for all concentratons studed (Fg. 4). Lght mcroscopy. Fg. 6 shows the shape change from bconcave to hghly transformed forms, whch occurs when FFA s added to red blood cells. The presence of 0.2 mm palmtc acd n a cell suspenson nduced the shrnkage of erythrocytes, appearance of monoconcave forms and cell aggregaton. At hgher effector concentratons cell swellng, membrane dsorderng and ghost formaton were observed. Dscusson Lght scatterng s a complex phenomenon that has found many uses n flow cytometry, partcularly n cell dscrmnaton. 129

8 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL 9..o- 1 0,39 0,37 + -o-3 ~., 0,35-~ ~ 0,33 9 r. 0,31 0,29 I 0,27 + 0,251 ~! ~ Ceffector ~ ~M 125 Fg. 5. Effect of palmtc acd (1), methyl ester of pahntc acd (2) and laurc aldehyde (3) on erythrocyte membrane mcrovscosty. PBS, ph 7.4, 18 ~ Vsser et al. [12] used red blood cells to demonstrate the senstvty of forward and perpendcular lght scatterng to erythrocyte shape. Dfferent ntrnsc factors and extrnsc agents can nduce red blood cell shape transformaton. Echnocytes are nduced by anonc or nononzed compounds and stomatocytes by catonc substances and non-penetratng anons [13]. Our flow cytometrc studes ndcate that FFA, ther dervatves as well as MDA sgnfcantly change the morphologcal propertes of erythrocytes. The effect of these hydrophobc agents depends on the chan length and the end group of the effector molecule. Red blood cells showed transent sze alteratons wth a reducton n dameter for the low effector concentratons and then an ncrease of cell dameter upon nteracton wth FFA. Cell shrnkage n the presence of palmtate, followed by erythrocyte swellng and haemolyss, s shown on mcrophotographs. The alphatc aldehydes and the methyl esters of FFA reduced the erythrocyte dameter. In the presence of hgh concentratons of methyl esters the formaton of two varous populatons of cells was observed as two peaks n the scatterng hstograms. The appearance of these two cell populatons can be due to a dfferent cell loadng wth the effector molecules. The number of cells wthn the fxed boundares (w/t) was smaller for LS2 (shape changes) than for LS 1 (sze changes) dagram n the presence of the effectors. 130

9 10 gm 10 gm C 10 u,m Fg. 6. Mcrophotographs of red blood cells n PBS: a - untreated cells, b - cells exposed to 0.2 mm of palmtc acd, c - cells exposed to 1 mm of palmtc acd. Dfferent effects of FFA and ther dervatves whch do not contan a charged carboxylc group, may be the result of an asymmetrc expanson of the outer or nner leaflets of the blayer n the presence of these effectors. Accordng to the blayer couple hypothess amphphlc compounds may affect the erythrocyte by ntercalatng preferentally nto one of the leaflets of the erythrocyte membrane [14]. It was proposed that amphphle-nduced shape changes n erythrocytes are ndcatve of the flp rate of the perturbng amphphle [15]. Sphaeroechnocytogenc amphphles nduced exovesculaton of the erythrocytes, whereas stomacytogenc amphphles nduced endovesculaton [3]. Recently t was shown that olec acd at a concentraton of 3.3 ~tm caused echnocytoss and the extent of transformaton was related to the effector molarty and locaton [13]. 131

10 FFA ncorporated nto the lpd blayer decreased the rotatonal dffuson rate of TMA-DPH whereas methyl esters of the FFA and correspondng alphatc aldehydes ncreased probe rotatonal moblty. Earler we showed [ 16] that FFA and ther dervatves ncreased the fludty of erythrocyte membranes as measured by pyrene excmer/monomer fluorescence ntensty rato. Ths dscrepancy can be due to the fact that the pyrene fluorescence rato reflects the lateral moblty of the probe and fluorescence ansotropy of TMA-DPH reflects rotatonal moblty or due to the dfferent probe localsaton n the membrane. An ncrease of erythrocyte dameter correlates wth a decrease of rotatonal moblty of the fluorescence probe ncorporated nto the lpd blayer whereas reducton of cell dameter s accompaned by an ncrease of probe rotatonal moblty. The morphologcal changes of the red blood cells are connected wth the membrane lpd blayer packng and moblty changes. The changes of cell sze are not connected wth the K + -efflux nduced by FFA and ther dervatves, but wth the drect membrane rearrangement n the presence of the effectors. Earler we showed that FFA, but not ther methyl esters or alphatc aldehydes, hghly ncreased membrane potassum permeablty [ 17]. The membrane modfcaton by MDA nduced sgnfcant changes n shape, sze and refractve propertes of erythrocytes and decreased the fludty of the erythrocyte lpd blayer. Earler we showed that ntra- and ntercrosslnkng of erythrocyte membrane proten molecules by MDA nduced structural changes of proten and lpd components of the membrane, manly the compact structure of spectrn [18]. We proposed that the effect of FFA and ther dervatves ncludes non-specfc nteractons wth the lpd blayer, leadng to a dsturbance of optmum complementarty between protens and surroundng lpds [16, 18]. It should be ponted out that varous cell and membrane parameters (erythrocyte shape, sze, stablty, permeablty) whch changed as the result of membrane rearrangement nduced by FFA and ther dervatves cannot be related to each other n a smple drect way. Ths study was supported by the Unversty of Ldd~ research grant No. 669/96. Dr. 1. Zavodnk s a recpent of the J6zef Manowsk Fellowshp Fund, Warsaw, Poland. References 1. Seeman P., Kwant W. O., Sauks T., Argent W. (1968) Bochm. B0phys. Acta 183, Isomaa B., Hagerstrand H., Paatero G., Engblom A. Ch. (1986)Bochem. Bophys. Acta 860,

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