Flip-Flop of Hydroxy Fatty Acids Across the Membrane As Monitored by Proton-Sensitive Microelectrodes ⁎ Elena E

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Biochimica et Biophysica Acta 1778 (2008) 1292–1297 www.elsevier.com/locate/bbamem

Flip-flop of hydroxy fatty acids across the membrane as monitored by proton-sensitive microelectrodes ⁎ Elena E. Pohl , Anna M. Voltchenko, Anne Rupprecht

Institute of Cell Biology and Neurobiology, Charité-Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany Received 12 October 2007; received in revised form 6 January 2008; accepted 31 January 2008 Available online 12 February 2008

Abstract

Hydroxyl group-containing fatty acids play an important role in anti-inflammatory action, neuroprotection, bactericide and anti-cancer defense. However, the mechanism of long-chain hydroxy fatty acids (HFA) transport across plasma membranes is still disputed. Two main hypotheses have been suggested: firstly, that protonated HFAs traverse across the membranes spontaneously and, secondly, that the transport is facilitated by proteinaceous carriers. Here, we demonstrate that the protonated HFA are able to move across planar lipid bilayers without protein assistance. This transport step is accompanied by the acidification of the buffer in receiving compartment and the pH augmentation in the donating compartment. The latter contained liposomes doped with HFA. As revealed by scanning pH-sensitive microelectrodes, the pH shift occurred only in the immediate vicinity of the membrane, while bulk pH remained unchanged. In concurrence with the theoretical model of weak acid transport, the pH value at maximum proton flux was almost equal to the pK of the studied HFA. Intrinsic pKi values were calculated from the electrophoretic mobilities of HFA-containing liposomes and were 5.4, 6.5, 6.9 and 6.3 for 2-hydroxyhexadecanoic, 16-hydroxyhexadecanoic, 12- hydroxydodecanoic and 9,10,16-trihydroxyhexadecanoic acids, respectively. © 2008 Elsevier B.V. All rights reserved.

Keywords: 2-hydroxyhexadecanoic acid; 16-hydroxyhexadecanoic acid; 12-hydroxydodecanoic acid; 9,10,16-trihydroxyhexadecanoic acid; Long-chain fatty acid; Proton flux; Bilayer membrane; Membrane permeability coefficient

1. Introduction (neuroprotectins) [2,3], bactericide and anti-cancer defence [4,5]. In animal tissues, 2-hydroxy fatty acids are frequently found in Saturated and unsaturated hydroxy fatty acids (further sphingolipids and have also been identified in skin. Although the addressed as HFA) play an important role in anti-inflammatory transport mechanism of these HFAs across plasma membranes is action (lipoxins, resolvins and protectins) [1], neuroprotection the key to understanding their involvement in regulating specific cellular responses, it has not yet been elucidated. A long standing controversy over whether long-chain fatty acids Abbreviations: BLM, bilayer lipid membrane; FA, long-chain fatty acids; (FA) in general exhibit fast transbilayer movement without protein HFA, long-chain hydroxy fatty acids; FH, protonated form of fatty acid; FA−, facilitation was addressed by Kamp and Hamilton [6], using pH as deprotonated form of fatty acid; USL, unstirred layer; PTFE, polytetrafluoroethy- a monitor of FA movement across vesicles. Our own studies with lene; DPhPC, Diphytanoyl-phosphatidylcholine; TES, N-[Tris(hydroxymethyl) planar membranes showed that pH changes are consistent with the methyl]-2-aminoethane-sulfonic acid; THPA, 9,10,16-trihydroxyhexadecanoic acid (9,10,16-trihydroxypalmitic, aleuritic acid); 12-HLA, 12-hydroxydodecanoic fast flip-flop of the un-ionized FA [7]. Nevertheless, controversies acid (12-hydroxylauric, sabinic acid); 16-HPA, 16-hydroxyhexadecanoic acid (16- persist, as exemplified by the disagreement about flip-flop of HFA. hydroxypalmitic, juniperic acid); 2-HPA, 2-hydroxyhexadecanoic acid (2-hydro- First reports on transbilayer movement were devoted to HFA as xypalmitic acid); PEG-PE, 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine- substrates for mitochondrial uncoupling proteins (UCP, [8–11]). N-[Nethoxy(Polyethyleneglycol) - 3000]; LUV, large unilamellar vesicles; SUV, Studying the acidification of the liposome interior using pH- small unilamellar vesicles; UCP, uncoupling proteins; Kow, octanol/water distribution coefficient; P, permeability coefficient sensitive fluorescent dye, Jezek et al. reported that several HFAs ⁎ Corresponding author. Tel.: +49 30450528141; fax: +49 30450528902. (named “inactive” fatty acids) were unable to move across the E-mail address: [email protected] (E.E. Pohl). lipid bilayer membrane spontaneously by the flip-flop mechanism

[10] and, thus, prevented the proton transport mediated by HFAs in chloroform-methanol (2:1) solution at a ratio of 60:40 mol%. Removal uncoupling proteins (UCPs) [11]. Because these HFAwere able to of the solvent by evaporation resulted in a thin lipid film on the wall of a round- bottom flask. An appropriate volume of buffer was added and the solution was partition into the lipid membranes, as detected directly using vortexed for 3 min. For the measurements of electrophoretical mobility, large HPLC [10], their inability to cross the membrane was attributed to unilamellar vesicles (LUVs) were prepared using a small-volume extrusion the low protonation probability. In contrast, other groups found apparatus (Avestin Inc., Ottawa, Canada). In order to obtain unilamellar vesicles that HFA derivatives were able to cross the membrane in their the solution containing multilamellar vesicles was extruded through a filter of protonated form [8,9]. 100 nm pore diameter [20] or sonicated. Both liposome types were added to planar bilayers. The maximal concentration of HFAs in vesicle suspension was In the present study we carried out steady-state measurements 0.77 mM/l, which does not exceed the concentration of free FA (FFA) in blood instead of the previously employed kinetic approach [8,10] to [21–23]. test whether the overall hypothesis [6] is consistent or (ii) the HFAs represent an exception. A HFA concentration gradient was 2.4. Scanning electrochemical microscopy generated across planar membranes and the resulting tiny pH shift was monitored in the immediate membrane vicinity by Proton transfer reactions due to permeation of the protonated FAs (Fig. 1,B) highly sensitive scanning microelectrodes [12]. The sensitivity shift the pH in the layer (unstirred layer, USL) adjacent to the membrane from its “ ” – bulk value. Through unstirred layers the transport of substances occurs solely by of 0.01 pH units was required because the Overton rule [13 diffusion [24]. The thickness of the USL on both sides of the membrane depends 16] predicts a lower membrane permeability for the more on the stirring conditions. The buffer was stirred on the cis- and trans-side of the hydrophilic hydroxy fatty acids than for the corresponding long membrane at equal velocity, which was kept constant throughout the experiment. pH shifts near the BLM were measured by a pH-microelectrode chain fatty acids. Taking into account the pKa shift of HFAs in the membrane environment, we carried out the experiments at at steady state conditions using the setup shown in Fig. 1, A and described in detail previously [12,25]. pH-microelectrodes comprised glass capillaries different pH. We examined several HFAs, whose ability to move (Science Products, Germany) containing a hydrogen ionophore cocktail spontaneously across the membrane is debated and showed that (Fluka, Germany). After pulling, their tips had a diameter of approximately their transmembrane movement is dependent on their hydro- 5 μm. The microelectrodes were moved at a velocity of 2 µm s− 1 perpendicular phobicity and can be described using a simple theoretical model to the surface of the membrane by a hydraulic microdrive manipulator applicable for all weak acids and bases [17]. (Narishige, Tokyo, Japan). The processes of BLM formation and pH- microelectrode movement were observed through the transparent window on the front side of the chamber by a microscope. 2. Materials and methods

2.1. Chemicals

Diphytanoyl-phosphatidylcholine (DPhPC, purity N99%) was obtained from Avanti Polar Lipids (Alabaster, USA). For the preparation of buffer solutions MES (2-Morpholinoethanesulfonic acid monohydrate), Tris (Tris-(hydroxyme- til)-aminometan), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), β-alanin (3-Aminopropionic acid) and KCl (all from Merck (Darmstadt, Germany)) were used. Fatty acids were obtained from Fluka Chemie AG, Buchs, Switzerland (9,10,16-trihydroxyhexadecanoic acid), Sigma Chemical Co., St. Louis, MO, USA (2-hydroxyhexadecanoic and 16-hydroxyhexadecanoic acids) and Sigma-Aldrich GmbH, Germany (12-hydroxydodecanoic acid). Fatty acids of the highest available purity were used. 1,2-Dipalmitoyl-sn-Glycero-3- Phosphoethanolamine-N-[Nethoxy(Polyethyleneglycol) - 3000] (PEG-PE) was purchased from Avanti Polar Lipids (Alabaster, USA).

2.2. Formation of planar bilayer membranes (BLM)

BLMs were formed either by the painting method [18] or by the monolayer apposition technique [19]. In the first case, lipid bilayers were formed in a hole 1–1.5 mm in diameter in the centre of a PTFE diaphragm dividing a chamber (Fig. 1, A) into two compartments. The membrane-forming solution contained 20 mg DPhPC in 1 ml of n-decane (Merck-Schuchardt, Munich, Germany). Using the monolayer apposition technique, membranes were formed by raising lipid monolayers on the top of two buffer solutions (1.5 ml per compartment) above the hole (0.2 mm in diameter) in the separating diaphragm. The hole in Teflon film (25 µm thick) was pre-treated with a hexane–hexadecane (95%:5%) Fig. 1. A. Experimental setup for pH shift measurements in the membrane mixture before the experiment. The membrane-forming solution contained vicinity by pH-sensitive microelectrodes. LUV- fatty acid-containing liposomes 20 mg DPhPC, dissolved in 1 ml of hexane (Aldrich Chemical Co., Alabama). (fatty acids are shown in black), BLM – planar bilayer membrane, ML – lipid The experiments were carried out at T=37 °C. monolayer, SB – stirrer bars, ME – microelectrode, RE – reference electrode, MD – microdrive, USL – unstirred layer, W – window for the membrane 2.3. Preparation of liposomes observation, A – amplifier, V – voltmeter, PC – personal computer. cis and trans indicate the respective sides of the chamber. The various parts of the HFA-containing liposomes were prepared (a) for measurements of setup are not drawn to scale. B. FA-mediated proton transfer reactions across the electrophoretical mobility and (b) for HFA delivery to planar bilayer membrane. Through unstirred layers (USL) the transport of substances occurs membranes. Therefore, DPhPC was dissolved in chloroform and mixed with solely by diffusion. 1294 E.E. Pohl et al. / Biochimica et Biophysica Acta 1778 (2008) 1292–1297

HFA-containing liposomes were added to the buffer solution at various concentrations (see figure legends) on the cis-side of the bilayer (Fig. 1, A). The reaction (FH↔FA− +H+; Fig. 1, B) on the trans-side caused a concentration gradient of protons within the USL. This gradient was measured in terms of a potential difference between the scanning pH microelectrode and a reference electrode, both placed in the buffer solution on the trans-side of the membrane. Experimentally measured H +-concentration profiles were employed to calculate the membrane permeability. The slope of these concentration profiles, which characterize the transport of the diffusing substances across the BLM, was obtained by a linear fit within a distance of 50 µm (the experimentally determined thickness of the USL) from the membrane surface. The proton flux, + JH, was estimated from the following Eq. (1) [26]:

Dbuff hDpH JH þ ¼ ð1Þ 2d0 where Dbuff is the diffusion coefficient of the buffer molecules; β, the capacity of the buffer solutions; ΔpH, the pH gradient on the BLM; δ0, the thickness of the unstirred − ⁎ 6 2 Fig. 3. The dependence of proton flux, JH +, across BLM on the concentration of layers in the vicinity of the BLM. The values Dbuff =4 10 cm /s (HEPES), 4.6⁎10−6 cm2/s (MES), 6.4⁎10−6 cm2/s (Tris) and 6.6⁎10−6 cm2/s (β-alanin) [12] 9,10,16-trihydroxyhexadecanoic (aleuritic) acid. Buffer contained 1 mM were used for the calculations. HEPES, 100 mM KCl, pH 7.2 (black circles) or pH 6.5 (grey circle). T=37 °C. Error bars represent the standard deviation of at least 5 experiments. 2.5. Measurements of the electrophoretical mobility of HFA-doped liposomes analysis implied that these HFAs should be permeable through the membrane. To estimate pK values of the HFAs studied, the electrophoretical mobility of HFA-reconstituted liposomes at different pH was measured using the Zetasizer Because the HFA transport was accompanied by proton Nano ZS (Malvern Instruments GmbH, Germany). Subsequently the corre- uptake and release, a pH shift appeared in the immediate sponding ζ-potentials were calculated as described previously [7,27,28]. membrane vicinity on both sides of the membrane. The resulting steady state pH profile was spatially resolved by microelectrode 3. Results measurements (Fig. 1, A and B). The proton profile formed within seconds of adding HFA-containing liposomes and did not 3.1. Measurements of HFA-induced proton transport across the change during the experiment. In Fig. 2, representative pH bilayer membrane profiles on the trans-side of the BLM at different concentrations of 12-hydroxydodecanoic acid in the buffer solution (100 mM Using scanning electrochemical microscopy, we investigated KCl, 1 mM MES) are shown. A proton concentration several fatty acid derivates (9,10,16-trihydroxyhexadecanoic acid, polarisation was found for all acids studied. It depended on the 12-hydroxydodecanoic acid, 16-hydroxyhexadecanoic, 2-hydro- fatty acid concentration used (see Fig. 3 for 9,10,16-trihydrox- xyhexadecanoic), whose ability to flip-flop across the membrane yhexadecanoic acid, Fig. 4 for 12-hydroxydodecanoic acid, data is disputed [8,10]. The octanol/water distribution coefficients of for other acids not shown). HFAs (Kow) were calculated using ALOGPS 2.1 Software (http:// www.vcclab.org, [29]). Log Kow for 12-HLA, 16-HPA, 2-HPA and THPAwere 3.43, 5.42, 5.70 and 2.83 respectively. The value

Fig. 4. (1) The dependence of proton flux across BLM on the concentration of 12-hydroxydodecanoic acid. HFA was added as constituent of liposomes (grey Fig. 2. Representative pH profiles, measured on the trans side of the BLM at circles). (2) Experiment as in (1); here the liposomes contained 40 mol% HFA, different concentrations of 12-hydroxydodecanoic acid added to the buffer 52.5 mol% DPhPC, 7.5 mol% PEG-PE-3000 (black circles). Buffer comprised solution (100 mM KCl, 1 mM MES, pH=5.5) on the cis side. Buffer solutions 1 mM MES, 100 mM KCl. Error bars represent the standard deviation of at least on the cis and trans sides were continually stirred with stirrer bars. 5 experiments. E.E. Pohl et al. / Biochimica et Biophysica Acta 1778 (2008) 1292–1297 1295

moving with the liposome as it moves through the solution. Assuming δ=0.2 nm [36,37], we obtain Φ=ζ, where ζ-potential is the parameter measured experimentally. C and k are the concentration of the electrolyte and the Boltzmann constant, respectively, e is the elementary charge, NA, the Avogadro number, z, the ion charge, T, the temperature, and ε, εo, the dielectrical constants. Because pH values in the vicinity of the charged lipid surface generally differ from that in the bulk phase, the maximal value of the Φs cannot be attributed to the ionization of all fatty acid molecules. To estimate the intrinsic pKa (pKi) of the HFA in a lipid bilayer, the following equation [38] was used (Eq. (5)): Φ Fig. 5. Dependence of surface potential, s, on buffer pH. DPhPC unilamellar A ¼ þ F s ð Þ liposomes were reconstituted with 9,10,16-trihydroxyhexadecanoic (aleuritic) pKi pKa : 5 acid. The buffer solution contained 20 mM KCl, 10 mM MES, 10 mM Tris, 2 303RT 10 mM β-alanin. where Φ s is the surface potential, and F, R, and Tare the Faraday constant, universal gas constant, and temperature, respec- Poorly soluble HFAs were added as constituents of liposomes tively. The experimental pH dependence of Φs was fitted as mimicking the FA delivery by albumin to the cell membranes. To shown in Fig. 5 for 9,10,16-trihydroxyhexadecanoic (aleuri- exclude possible alterations in the membrane permeability due to tic) acid. The pKi estimated are equal to 5.4, 6.5, 6.9 and fusion events between the planar membrane and liposomes, 6.3 for 2-hydroxyhexadecanoic, 16-hydroxyhexadecanoic, PEG-PE–which is known to prevent the fusion events [30,31]– 12-hydroxydodecanoic and 9,10,16-trihydroxyhexadecanoic was also added to either the liposomes (Fig. 4) or the bilayer acid respectively. membrane (data not shown). Similar concentration dependences in the absence and in the presence of PEG-PE demonstrated that 3.3. Estimation of proton flux at different pH values of buffer no artifacts arising from proton leak by fusion of vesicles with solution planar membranes occurred. The pH shift at the membrane–water interface induced by 3.2. Estimation of fatty acid pK using the electrophoretic the flip of HFAs was expected to depend on the pH value of the mobility of HFA-containing liposomes buffer solution as described for FA with a chain length of C6– C12 [17]. The proton flux in the presence of 300 µM of 9,10,16- The extremely poor solubility of long-chain FAs, in general, and trihydroxyhexadecanoic acid increased from 3.6 pmol/cm2sat HFAs, in particular, causes difficulties in determining their pK, both pH 7.2 to 47 pmol/cm2s at pH 6.5 (Fig. 3). As demonstrated in in water solution and in the lipid phase. The reported values for FAs Fig. 6, at different concentrations of 12-hydroxydodecanoic ranged between 5, 7.5 and 10.15 [32–34]. The latter value was acid, the proton flux reached a maximum at a pH near the pKi found by titration of the solution with HCl after stearic acid was value of the FA in lipid. Experimental results were fitted (solid dissolved at pH=10.5 [33]. In our previous work [7], we estimated the pK of stearic acid from pH-dependent surface potential changes of reconstituted liposomes. The estimated pK of 7.5 was in agreement with the pK of stearic acid reported by Hamilton et al. [32]. Because the pK values of 9,10,16-trihydroxyhexadecanoic acid, 12-hydroxydodecanoic and 16-hydroxyhexadodecanoic FAs had not as yet been reported in the literature, we estimated these values in the present work by surface potential (Φs)measurements. Φs values were calculated from the electrophoretical mobility of liposomes according to the Eqs. (2)–(4) [7,27,35]: 2kT 1 þ aexp½jd A ¼ ln ð2Þ ze 1 aexp½jd exp½zeA =2kT 1 a ¼ s ð3Þ exp½þzeAs=2kT 1 sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2e2z2CN j ¼ A ð Þ Fig. 6. The experimental (symbols) and theoretical (solid line) dependencies ee 4 0kT of proton membrane flux, JH+, on buffer pH. HFA-containing liposomes (12-hydroxydodecanoic acid) were added to the cis-side of the membrane in where δ is the thickness of an imaginary sphere around the a concentration of either 99 µM (black circles) or 117 µM (grey triangles). liposome, called the “shear plane”, which limits the solvent Buffer solution contained 1 mM MES, 1 mM Tris, 100 mM KCl. 1296 E.E. Pohl et al. / Biochimica et Biophysica Acta 1778 (2008) 1292–1297

lines in Fig. 6) using the theoretical pH dependence of JH+ acids exhibit similar flip-flop rates [45] despite large differences (Eqs. (6) and (7)) [17]. in their partition coefficients (dodecanoic, log Kow =4.98; ÀÁ tetradecanoic, log Kow =5.84; hexadecanoic, log Kow =6.87; þ ¼ M USLa= ðÞþ a USLa= þ M ð Þ JH C0PFH PFA 21 PFA 2 PFH 6 octadecanoic, log Kow =7.83, as calculated in this paper). Because of their different partitioning behaviour, variation in PFH among a ¼ 10pHpK ð7Þ the HFAs was also expected. The presented data (Figs. 3 and 4) indicate that ~10× higher concentrations of 9,10,16-trihydrox- С M where 0 is the FA concentration in the buffer solution, PFH , the yhexadecanoic acid (3 OH groups) are needed to obtain the same permeability coefficient of the protonated FA form (FH) across H+ flux as with 12-hydroxylauric acid (1 OH group), which is USL USL the membrane, and P and P , the permeability coefficients apparently a consequence of partitioning (the log Kow for 9,10,16- FH FA − of the protonated (FH) and deprotonated (FA ) forms through trihydroxyhexadecanoic and 12-hydroxylauric acid were 2.83 the unstirred layer (USL), respectively. PFH of 12-hydroxydo- and 3.43 respectively). decanoic acid across the membrane estimated from the fitted To decide whether HFAs are “active” or “inactive”, their ⁎ − 5 ⁎ − 5 experimental data was equal to 1.9 10 ±0.8 10 cm/s. translocation (flip-flop) rate constants (kHFA) must be compared − The maximal JH+ occurs at a pH close to the pKi of HFAs in with the turn over rate of a FA transporter e.g. uncoupling lipid membranes (Figs. 2, 6). This result is not surprising, if one protein (UCP, [46,47]). The term “inactive FA” is only justified, bears in mind that the measurements are carried out in steady if facilitated transport is faster than simple diffusion. kHFA for the state conditions and the concentration of protonated and protonated fatty acid can be estimated as the ratio of proton flux deprotonated FA forms should be equal at pH=pK. The strong JH+ and HFA surface concentration [48]. For example, for 12- sensitivity of HFA transport to pH indicates the importance of hydroxydodecanoic acid the surface concentration of the the regulatory function of pH on FA cell uptake [6,7,39,40]. protonated HFA at pH=pK (50% ionization) under considera- − 12 tion of the partitioning coefficient Kow was about 9×10 mole 4. Discussion cm− 2. With respect to the HFA-flux determined by scanning electrochemical microscopy (Fig. 6), the translocation rate − 1 Using the highly sensitive technique of scanning electro- constant, kHFA, is equal to kHFA =13 s . Given the several chemical microscopy [12,41,42], we demonstrate that the sources of error, e.g. omitting buffer effects (s. above), this kHFA transport of 9,10,16-trihydroxyhexadecanoic, 12-hydroxydode- is only a rough estimate of the lower limit. Similar conclusions canoic FAs (reported to be “inactive”), as well as 2-hydro- were drawn for all other HFA studied. Since the turn over rate xydodecanoic and 16-hydroxyhexadodecanoic FAs (supposed calculated for UCP2 in the presence of the most potent UCP to be “active”) is accompanied by proton uptake at one water/ activator, arachidonic acid, is about 4 s− 1 a protein-mediated lipid interface and proton release at the opposite interface, thus facilitation of protonated HFA transport can be excluded. proving that all long-chain hydroxy fatty acids (HFAs) studied flip from one side of the membrane (cis) to the other (trans). This Acknowledgements contradicts an earlier report [10] but is in accordance with the observation that substitution of the FA methyl group for This work was supported by the German Research Founda- hydrophilic groups, such as hydroxy group, still permits the tion (DFG: Po 524/2-1, Po 524/2-2). The authors thank Dr. P.Pohl measurement of proton uptake in liposomes [8,9,43]. for the helpful discussion, P. Douglas and J. A. Liebkowsky for Following the “Overtone rule” [14,44], we expected the the editorial assistance. membrane permeability of HFA to be lower than the permeability of their non-hydroxylated counterparts, because HFAs are more References soluble in water and partition into the lipid membrane to a smaller ⁎ -4 extent. Accordingly, PFH of 0.2 10 cm/s was estimated for the [1] J.M. Schwab, C.N. Serhan, Lipoxins and new lipid mediators in the resolution of inflammation, Curr. Opin. Pharmacol. 6 (2006) 414. protonated 12-hydroxydodecanoic acid (log Kow =3.43) from the fit to the experimental data. It is several orders of magnitude lower [2] P.K. Mukherjee, V.L. Marcheselli, C.N. Serhan, N.G. Bazan, Neuropro- tectin D1: a docosahexaenoic acid-derived docosatriene protects human than the apparent PFH of 1.61 cm/s for palmitic acid [13] (log retinal pigment epithelial cells from oxidative stress, Proc. Natl. Acad. Sci. Kow =6.87). However, it should be taken into account that U. S. A. 101 (2004) 8491. omitting buffer effects from calculations always results in an [3] V.L. Marcheselli, S. Hong, W.J. Lukiw, X.H. Tian, K. Gronert, A. Musto, M. Hardy, J.M. Gimenez, N. Chiang, C.N. Serhan, N.G. Bazan, Novel underestimation of membrane permeability. The actual PFH may exceed the estimated P by at least one order of magnitude docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infil- FH tration and pro-inflammatory gene expression, J. Biol. Chem. 278 (2003) [12,41]. According to the data reported by Evtodienko et al. [17], 43807. the permeability coefficients of short-chain fatty acids with [4] A. Abe, K. Sugiyama, Growth inhibition and apoptosis induction of hu- comparable hydrophobicities as octanoic (log Kow =2.84) and man melanoma cells by omega-hydroxy fatty acids, Anticancer Drugs 16 (2005) 543. decanoic acids (log Kow =3.91) are equal to 0.17 and 0.22 cm/s, respectively. Whether long-chain FA would fit into the experi- [5] I. Shureiqi, W. Jiang, X. Zuo, Y. Wu, J.B. Stimmel, L.M. Leesnitzer, J.S. Morris, H.Z. Fan, S.M. Fischer, S.M. Lippman, The 15-lipoxygenase-1 mentally determined dependence of permeability on the partition product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-delta to coefficient is unclear [44]. Using a kinetic fluorescence-based induce apoptosis in colorectal cancer cells, Proc. Natl. Acad. Sci. U. S. A. approach, it has been shown, that a variety of long-chain fatty 100 (2003) 9968. E.E. Pohl et al. / Biochimica et Biophysica Acta 1778 (2008) 1292–1297 1297

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