Differential effects of conjugated linoleic acid isomers on the biophysical and biochemical properties of model membranes

Abstract

Conjugated linoleic acids (CLA) are known to exert several isomer-specific biological effects, but their mechanisms of action are unclear. In order to determine whether the physicochemical effects of CLA on membranes play a role in their isomer-specific effects, we synthesized phosphatidylcholines (PCs) with 16:0 at sn-1 position and one of four CLA isomers (trans 10 cis 12 (A), trans 9 trans 11 (B), cis 9 trans 11 (C), and cis 9 cis 11 (D)) at sn-2, and determined their biophysical properties in monolayers and bilayers. The surface areas of the PCs with the two natural CLA (A and C) were similar at all pressures, but they differed significantly in the presence of cholesterol, with PC-A condensing more than PC-C. Liposomes of PC-A similarly showed increased binding of cholesterol compared to PC-C liposomes. PC-A liposomes were less permeable to carboxyfluorescein compared to PC-C liposomes. The PC with two trans double bonds (B) showed the highest affinity to cholesterol and lowest permeability. The two natural CLA-PCs (A and C) stimulated lecithin-cholesterol acyltransferase activity by 2-fold, whereas the unnatural CLA-PCs (B and D) were inhibitory. These results suggest that the differences in the biophysical properties of CLA isomers A and C may partly contribute to the known differences in their biological effects.

Fig. 1

a) Surface area isotherms of CLA-PCs and control (unconjugated 18:2) PC. Compressions were performed at ambient temperature (~24 °C) on a surface of purified water at a rate of 10 cm²/min. Isotherms are representative of at least 3 runs; error ranges were ± 3 Ų molecule⁻¹. b) Condensation of CLA-PCs and control PC monolayers by cholesterol at 20 mN/m. Data for condensation were derived from the pressure-area curves similar to those shown in Fig. 1a, as described in Materials and Methods.

Fig. 1

a) Surface area isotherms of CLA-PCs and control (unconjugated 18:2) PC. Compressions were performed at ambient temperature (~24 °C) on a surface of purified water at a rate of 10 cm²/min. Isotherms are representative of at least 3 runs; error ranges were ± 3 Ų molecule⁻¹. b) Condensation of CLA-PCs and control PC monolayers by cholesterol at 20 mN/m. Data for condensation were derived from the pressure-area curves similar to those shown in Fig. 1a, as described in Materials and Methods.

Fig. 2. Effect of CLA-PC isomers on the transition temperatures (T_m) of dipalmitoyl (16:0–16:0) PC

Multilamellar liposomes of 16:0–16:0 PC (2 mg/ml) alone or containing 10 mol% or 25 mol% of CLA-PC or LA-PC (control) were prepared in distilled water and at least 4 heating scans were performed in Microcal VP-DSC calorimeter. The transition temperatures (T_m) were determined from the fourth scan.

Fig. 3. Cholesterol-binding properties of CLA-PCs

The ability of the liposomes prepared from each of the CLA-PCs to extract labeled cholesterol from [¹⁴C]-cholesterol-β-methyl cyclodextrin complex was determined as described in the text, in the absence and presence of 20 mol% egg SM. The amount of cholesterol extracted by control LA-PC (16:0–18:2, unconjugated) was taken as the baseline, and all other values were expressed as % above or below that value. The values are shown are mean ± S.D of 3–5 experiments. * p< 0.05, and ** p<0.005 compared to LA-PC alone. † p<0.005, compared to LA-PC + 20 mol% SM.

Fig. 4. Cholesterol-binding properties of isomers of 16:0–18:1 PC

PCs containing the different isomers of 18:1 at sn-2 position were chemically synthesized, and their affinity to cholesterol was determined as described in the text. The values are expressed as % of the value obtained with 16:0–18:2 (unconjugated) PC, and are average of two separate experiments. The n-3 trans isomer of 18:1 free fatty acid was not available, and therefore is not included in the study.

Fig. 5. Carboxyfluorescein permeability of CLA-PC liposomes

Small unilamellar vesicles of various CLA-PCs, containing the trapped carboxyflurescein were prepared by sonication, and the untrapped dye was removed by gel filtration chromatography. The leakage of the dye following its dilution with excess buffer was monitored in a flurometer as described in the text. The values shown are mean ± S.D of 4 separate experiments, and are expressed as % of the values obtained with unconjugated 16:0–18:2 PC.

Fig. 6. Fluorescence anisotropy values of DPH-PC incorporated into various PC liposomes

DPH-PC was incorporated into the liposomes at probe: PC ratio of 1:200, and fluorescence anisotropy was determined at various temperatures.

Fig. 7. Oxidation of various PC species in presence of 1 mM AAPH

PC liposomes (0.1 μmol PC) were incubated at 37 °C in presence of 1 mM AAPH. At the indicated period of time the total lipids were extracted and the fatty acid composition analyzed by GC, after adding 17:0–17:0 PC as internal standard. The % CLA remaining was calculated, relative to 17:0. The data shown are averages of 3 separate experiments.

Fig. 8. Effect of CLA on LCAT activity

Proteoliposome substrates containing the respective PCs and labeled cholesterol were prepared as described in the text, and incubated with a semi-purified preparation of human LCAT for 30 min. The percent of labeled cholesterol esterified was determined after TLC separation of the lipid extract. The values shown are mean ± S.D of 3 separate assays.

Fig. 9. A postulated mechanism for the increased binding of cholesterol by t10c12 CLA-PC

It is proposed that position of the first cis double bond (starting from the carboxy end) is critical in cholesterol binding. Because of the kink in the acyl chain generated by the presence of the cis double bond, the pocket formed by the cis 12 double bond in t10c12 CLA-PC is larger than the pocket formed by the cis 9 double bond in c9t11 CLA-PC, and therefore more easily accommodates the bulky and rigid sterol ring. Furthermore, in a membrane that contains a saturated fatty acid at sn-1 and t10c12 at sn-2, the cholesterol may interact with both sides of the PC molecule, whereas it interacts only with the saturated acyl group in the PC containing c9t11 CLA. Since the trans double bond does not introduce a kink in the acyl chain, it is considered to be equivalent to a single bond in this model.