Effect of α-tocopherol incorporation on glucose permeability and phase transition of lecithin liposomes

Chemistry and Physics of Lipids, 23 (1979) 13-22 © Elsevier/North-Holland Scientific Publishers Ltd.

EFFECT OF c~-TOCOPHEROL INCORPORATION ON GLUCOSE PERMEABILITY AND PHASE TRANSITION OF LECITHIN LIPOSOMES KEN J! FUKUZAWA, HIROHIKO IKENO, AKIRA TOKUMURA and HIROAKI TSUKATANI Faculty of Pharmaceutical Science, University of Tokushima, Shomaehi, Tokushima (Japan)

Received April 21st, 1978

accepted June 9th, 1978

Liposomes were prepared from dipalmitoyUecithin, dimydstoyllecithin, dioleoyUecithin, egg lecithin, and soybean lecithin, and the effects of incorporation of various quantities of a-tocopherol or its analogs on permeability of the liposomes to glucose were studied at various temperatures (4-40°C). Results showed that increase in the quantity of a-tocopherol incorporated into dipalmitoyUecithin and dimyristoyilecithin liposomes lowered the transition temperature for marked release of glucose and also decreased the maximum rate of temperature-dependent permeability, a-Tocopherol also had similar but less marked effects on the permeability of dioleoylleeithin and egg lecithin liposomes, but little effect on those of soybean lecithin, which has a higher degree of unsaturation. In dipaimitoyUecithin liposomes phytol showed a similar effect on permeability to that of a-tocopherol, but phytanic acid caused a different pattern of temperature-dependent permeability. With analogs of a-toeopherol, the regulatory effect on permeability decreased with shortening and disappearance of the isoprenoid side chain. The significance of these observations is discussed in relation to the physiolo#eal functions of tocopherols in natural membranes.

I. Introduction It is well known that tocopherol (vitamin E) is localized in cellular membranes where it is biologically active. Deficiency o f vitamin E induces alterations in the structure and function o f biomembranes. Tocopherol has generally been considered to prevent membrane deterioration by virtue o f its lipid-antioxidant properties. However, recent findings have indicated that the amphiphilic structure o f tocopherol, and especially the physical properties o f its phytyl side chain are important for membrane stabilization [ 1,2]. The importance of the fluidity o f membrane lipids in regulating membrane functions has been suggested by a number of recent studies [ 3 - 5 ] . It has also been suggested that a-tocopherol functions in controlling the fluidity o f phospholipids in membranes [6,7]. The condensing and fluidizing effects o f tocopherol in model membrane systems have been studied by a variety o f techniques, such as monolayer studies [2,8], nuclear magnetic resonance [9] and fluorescence spectroscopy [ 10]. This paper reports the effects o f incorporation of a-tocopherol and its analogs on membrane structure and fluidity, as evidenced by changes in permeability of phospholipid vesicles to glucose. The possible structural role o f vitamin E in the membranes is discussed. 13

14

K. Fukuzawa et al., Tocopherol and lecithin liposomes

II. Materials and Methods A. Chemicals and enzymes

The chemicals and enzymes used were purchased from the following companies: Sigma Chemical Co., St. Louis, MO (dipalmitoyUecithin, dimydstoyllecithin, dioleoyllecithin, soybean lecithin and dicetyl phosphate); Kyowa Hakko Kogyo Co., Tokyo (ATP); Oriental Yeast Co., Tokyo (hexokinase, glucose-6-phosphate dehydrogenase and NADP). DL-a-Tocopherol and its analogs (as shown in Table 1) were kindly supplied by Dr. S. Kijima, Eisal Co., Tokyo. Egg lecithin was prepared in our laboratory by the method of Pangborn [ 11 ]. The purity of all lecithins used was checked by thin-layer chromatography on silica gel plate with chloroform/methanol/water (65 : 35 : 5, by vol.) as solvent. The fatty acid species and compositions of egg lecithin and soybean lecithin (as shown in Table 2) were determined by gas chromatography. After being prepared by methanolysis in acid-methanol [12], fatty acid methyl esters from phosphollpids were separated and quantified by a Shimadzu gas chromatograph GC-6A equipped with a column (3 mm × 1.5 m) packed with 10% EGSP on 60-80 mesh Chromosorb WAW. The column oven temperature was 170°C and the nitrogen flow rate was 60 ml/min. Tocopherol and its analogs used in the present study gave a single spot on thin-layer chromatography with cyclohexane/ether (80 : 20, by vol.). Stock solutions of the phospholipids, dicetyl phosphate and tocopherol analogs were prepared in chloroform and stored at --20°C. Concentrations of phospholipids were determined by the method of Chalvardjan and Rudnicki [ 13] for total phosphate. B. Preparation o f liposomes and measurement o f permeability to glucose

Liposomes were prepared by a slight modification of the method of Demel et al. Table 1 Chemical Structures Name

Polar head group

Hydrophobic side chain (R)

TMC-O

HO~~

-CH3

TMC-I "I"]~¢~C-3

(c¢-Tocopherol) Phytol

Phytanic acid

CH3

CH~

H3 cH3

2,5 ,7,8-tetramethylehro~an-6-ol HOH2C~ _ _

CH3

/~/Cfl 3 Hooc - , .

~H~

-CH2CHzCH2CHCH3 CH3 "(CH2CH2CHa~H) 3CH3 CH3 CH3 -(CH2CH2CH2~H)3

CH3

-(CHzCHzCH2~H)3CH3

K. Fukuzawa et al., Tocopherol and lecithin liposomes

15

Table 2 Fatty acid compositions of egg lecithin and soybean lecithin Fatty acids

Palmitic acid Palmitoleic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Arachidonic acid

Composition (%) egg lecithin

soybean lecithin

38.9 1.3 10.8 33.4 13.4 ND 2.2

10.7 NDa 3.5 6.7 72.6 6.5 ND

aND: not detected. [14]. In this procedure, 2/amol of lecithin, 0.2/~mol of dicetyl phosphate and appropriate quantities of tocopherol analogs in the desired molar ratios were put into a I0 ml conical flask. After removal of the chloroform under reduced pressure, 0.2 ml of 0.3 M glucose was added to the dried lipid film, and the f'tim was dispersed in a vortex mixer under a nitrogen atmosphere to give a final lecithin concentration of 10/~mol/ml. Most untrapped glucose was removed by dialysis against 200 ml of 0.15 M NaCI solution. The reaction was started by adding 5/el of liposome preparation to 1 ml of assay mixture (50 mM Tris-buffer, pH 7.5,107 mM NaC1, 1.75 mM MgCI2, 75 taM CaC12, 1 mM ATP, 0.5 mM NADP, 2 units ofhexokinase and 1 unit of glucose-6-phosphate dehydrogenase). The amount of glucose released was estimated by measuring the absorption at 340 nm. The total amount of glucose trapped in liposomes was determined by adding Triton X-100. Enzyme activities in the experimental conditions did not vary significantly over the temperature range C4-40°C) used.

III. Results and discussion

DipalmitoyUecithin liposomes with and without a-tocopherol were incubated at various temperatures for 10 min (fig. 1). The amount of glucose trapped in dipalmitoyllecithin liposomes was 2.46 mol per mol phosphorus of lipid. Liposomes derived from dipalmitoyUecithin and dicetyl phosphate released little glucose marker when incubated below 35°C. In the temperature range (38-40°C) corresponding to the phase transition temperature of 4 I°C [ 15], the permeability to glucose became much higher (fig. 2(a)). Dirnyristoyllecithin liposomes also showed a maximum diffusion rate at near the temperature of phase transition (23°C) (fig. 2(b)). Incorporation of a-tocopherol into dipalmitoyUecithin and dimyristoyllecithin liposomes affected their

16

K. Fukuzawa et al., Tocopherol and lecithin liposomes 50

4o°c .0-

40

3O 33"C

~ 2o Qo

4o*c 33"C 10

0 I n c u b a t i o n time

(min)

Fig. 1. Time course of glucose release from liposomes of dipalmitoyllecithin with and without a-toeopherol. Samples of about 5 t~l of liposome preparations were incubated at 33° or 40°C in cuvettes containing 1 ml of Tris-buffer (pH 7.5) with enzymes and cofactors, and changes of absorbance at 340 nm were measured for 10 rain. Liposome composition (molar ratios): (o) dipalmitoylleeithin, dicetyl phosphate (10 : 1); (e) dipalmitoyllecithin, dicetyl phosphate, e-tocopherol (10 : 1 : 2).

temperature-dependent permeabilities to glucose: with increase in the molar ratio of a-toeopherol incorporated the temperature ranges in which marked leakage of glucose occurred were progressively lowered (fig. 2). Schmidt et al. [10] demonstrated in ESR studies on liposomes using spin label TEMPO (2,2,6,6,-tetramethylpiperedine-l-oxyl) that increase in th incorporation of a-tocopheryl acetate into membranes induced a proportional decrease in the phase transition temperature and broadening of the temperature range of phase separation. 50

*

Q;

O0

(a)

(b)

40

.

3o

c~

SO

60

4O

20 m o u

o

~ 2O

10

20

30 Temperature

40 ( °C )

0

2'0

30 Temperature ( °C )

I0

40

Fig. 2. Temperature dependence of glucose release from liposomes of (a) dipalmitoyUecithin and

(b) dimyristoyllecithin with and without a-tocopherol or TMC-O (dotted line) on incubation for 10 rain. Liposome composition (molar ratios): (o) lecithin, dicetyl phosphate (10 : 1 ); (.) lecithin, dicetyl phosphate a-tocopherol (100 : 1 0 : 5 ) ; (A) lecithin, dicetyl phosphate, a-tocopherol (10 : 1 : 1); (v) lecithin, dicetyl phosphate, ce-tocopherol (100 : 10 : 15); (e) lecithin, dicetyl phosphate, a-toeopherol (10 : 1 : 2); (o) lecithin, dicetyl phosphate, TMC-O (10 : 1 : 2).

K. Fukuzawa et al., Tocopherol and lecithin liposomes

17

The maximum rates of temperature-dependent permeability of dipalmitoyllecithin and dimyristoyllecithin liposomes decreased with increase in ct-tocopherol incorporation. Cholesterol is present in many natural membranes; when less than 30 mol% cholesterol was incorporated into dipalmitoyllecithin liposomes it also lowered the phase transition temperature, but markedly increased the maximum permeability to glucose [16]. As shown in fig. 3, increase in the quantity ofct-tocopherol incorporated progressively increased glucose release at 33°C but decreased at 40°C. This decrease in permeability at 40°C caused by a-tocopherol could be explained as due to decrease in me mobility of the acyi chain of lecithin, because molecular models have shown that the methyl groups of the side chain of a-tocopherol reduce the mobility of the fatty acyl chain by steric interaction. Moreover, Schmidt et al. [10] have reported that the presence of 28 tool% tocopheryl acetate results in a decreased lateral diffusion coefficient for pyrene and an increased order of fatty acid chains in dipalmitoyllecithin membranes above phase transition temperatures. At 37°C, ct-tocopherol showed a biphasic action on permeability to glucose depending upon its concentration;it caused increased permeability at a concentration below 5 mol% and decreased permeability at a concentration above 5 mol%. We have previously reported that a-tocopherol has a biphasic effect on lysosome membranes, causing stabilization at a concentration of 10-4 M and labilization at a higher concentration of 5 × 10-4 M [ 17]. A considerable quantity of dipalmitoyllecithin has been demonstrated in membranes, and assuming a non-random distribution in the membrane, a local liquidifying effect of a low concentration of tocopherol at physiological temperature cannot be excluded.

50 4O v

30 o

20 Q;

~ IO

o

o.1

0.2

Molar ratio (~-Tol/DPL)

Fig. 3. Effect of incubation temperature on the permeation rate of glucose through dipalmitoyllecithin liposomes containing various quantities of a-tocopherol. The incubation temperatures were as follows: (o) 40°C; (o) 37°C; (a) 33°C; (A) 25°C.

18

K. Fukuzawa et aL, Tocopherol and lecithin liposomes

Cholesterol molecules interact strongly with molecules containing oleoyl residues [18]. However, it has been proposed that ct-tocopherol interacts primarily with polyunsaturated phospholipids rather than with phospholipids containing a monoene acyl residue, because it caused more increase in surface pressure when it penetrated into monolayers of polyunsaturated phospholipids than when it penetrated into those of monounsaturated or saturated ones [8]. Liposomes were prepared from dioleoyllecithin, egg lecithin and soybean lecithin to determine how the substitution of saturated fatty acids for unsaturated ones influences the temperature dependence of permeability with and without incorporated a-tocopherol (fig. 4). It has been reported that the permeability to a marker (glucose) of liposomes prepared with egg lecithin or synthetic 1-stearoyl-2-oleoyUecithin was greater at 2-5°C than at 20-30°C, and that the permeability increased again above 30°C [19]. Similar results were obtained in the present study (fig. 4(a)): on incubation for 20 min, above 50% of the trapped glucose was released at 40°C but only a few percent was released at 10-30°C. When a-tocopherol was incorporated into liposomes prepared with egg lecithin significantly less glucose was released. Decrease in the length of the isoprenoid side chain reduced the effect on permeability to glucose. The ability of a-tocopherol to decrease permeability to glucose depending on the length of the phytyl side chain, correlated well with its activity to stabilize lysosomal membranes and capability to expand fatty acid

,""I

60 (b) 50

,)

Co)

" "

50

f~i'540 ;'

~ m o

/J

30

////

2O

q

/

~"

!~/) ~2o •

~1o i

i

0

10 20 30 Temperature ( °C )

40

0

10

20

Temperature

30

40

( °C )

Fig. 4. (a) Temperature dependence of glucose release from liposomes of egg lecithin with and without c~-tocopherol or TMC-1 (dotted line) on incubation for 20 rain. (b) Effect of incorporated c~-tocopherol on the temperature-dependent permeability to glucose of liposomes prepared with dioleoylleeithin (solid line) and soybean lecithin (dotted line). Glucose release from liposomes was assayed after 30 rain incubation at various temperatures. Liposome composition (molar ratios): (v) lecithin, dicetyl phosphate (10 : 1); (A) lecithin, dieetyl phosphate, a-tocopherol (10 : 1 : 1); (e) lecithin, dieetyl phosphate, ~-toeopherol (10 : 1 : 2); (Q) lecithin, dicetyl phosphate, TMC-1 (10 : 1 : 2).

If. Fukuzawa et aL, Tocopherol and lecithin liposomes

19

monolayers [2]. Contrary to our results, Cushley and Forrest [9] demonstrated by 3~P-NMR studies, using a lanthanide-induced shift technique, that incorporation of c~-tocopherol or phytanic acid increased the permeability of egg lecithin bilayers to Pr 3+. The reason for this discrepancy is ur&nown, but it must be caused by differences in the experimental methods and conditions used, such as the kind of vesicles (multilamellar liposomes or small liposomes prepared by sonication), the nature of the marker (glucose or Pr 3÷) and/or the membrane composition (the presence or absence of the charged amphiphathic compound, dicetyl phosphate). As fig. 4(b) shows, liposomes prepared with dioleoyUecithin or soybean lecithin could trap glucose even through their phase transition temperatures were below 0°C. Their permeability also showed strong temperature dependence, increasing with increase of temperature. The slope of the temperature-dependent curves of the permeation rates through dioleoyllecithin and soybean lecithin liposomes were much less than those of dipalmitoyllecithin and dimydstoyllecithin liposomes. When a-tocopherol was incorporated into dioleoyllecithin liposomes, it decreased the temperaturedependent permeability in proportion to its concentration at all temperatures tested (4-40°C). It had a similar but smaller effect on the permeability of soybean lecithin liposomes. These observations are in agreement with our previous results on monolayers [2] : that is, a-tocopherol-induced increase in the surface pressure of fatty acid films decreased significantly with increase in the number of unsaturated bonds in the paraffin chain. Diplock et al. [20] indicated that the ability of a-tocopherol to decrease the permeability depends on a content of arachidonic residues in the phospholipid (the permeability is inhibitied more strongly when the phospholipid had a higher content of arachidonic residues). Cholesterol is reported to cause significant reduction of permeability to glucose of egg lecithin liposomes and slight reduction of the permeability of dilinoleoyllecithin liposomes [19]. Recently, Albarracin et al. [2 I] found that skeletal muscle of vitamin E-deficient rabbits has an increased content of cholesterol, which may perhaps compensate in part for the lack of vitamin E. The similarities in the effects of tocopherol and cholesterol on the permeabilities of unsaturated lipid bilayers and the stabilities of biomembranes indicate that cholesterol may function as a partial substitute for tocopherol. Phytol had a similar effect to a-tocopherol on the permeability of dipalmitoyllecithin liposomes (fig. 5), while TMC-0, an a-tocopherol analog without the isoprenoid side chain, had no influence (fig. 2(a)). Thus the effect of a-tocopherol on the permeability of lecithin membranes appears to require the phytyl side chain rather than the hydrophylic chroman ring. Maggie et al. [8] found in studies on monolayers that the optimal interactions between tocopherols and phospholipid molecules occur when the lengths of their hydrophobic moieties are approximately equal. Leibowitz and Johnson [22] reported that there is no correlation between the antioxidant effect of tocopherol and its effect in preventing loss of cations trapped in liposomes. Lucy and Dingle [1] also observed that in vitro hemolysis by added retinol is inhibited by a low concentration of a-tocopherol, or of other isoprenyl compounds, such as phytol,

20

K. Fukuzawa et al., Tocopherol and lecithin liposomes

60 50

---'e'~

"'-

b,

40 O;

~ 30 ~ 2o O

g

lO

20 Temperature

30

40

( *C )

Fig. 5. Temperature dependence of glucose permeability through liposomes prepared with dipalmitoylleeithin containing phytol or phytanic acid (dotted line). The liposomes were incubated under the same conditions as for Fig. 2(a). Liposome composition (molar ratios): (o) dipalmitoyllecithin, dieetyl phosphate (10 : 1); (~) dipalmitoylleeithin, dicetyl phosphate, phytol (100 : 10 : 5); (A) dipalmitoyllecithin, dieetyl phosphate, phytol (10 : 1 : 1); (s) dipalmitoyUecithin, dicetyl phosphate, phytol (10 : 1 : 2);(v) dipalmitoylleeithin, dicetyl phosphate, phytanic acid (10 : 1 : 1); (e) dipalmitoylleeithin, dieetyl phosphate, phytanie acid (10 : 1 : 2).

squalene, ubiquinone.30 and vitamin K~, while antioxidants, such as N,N'.diphenyl-p. phenylenediamine and hydroquinone, had no effect. This activity was thought not to be caused by the redox system of tocopherol, but rather by the physical interaction of the isoprenoid side chain with fatty acid residues of phospholipid molecules in the hydrophobic interior of the membranes. Incorporated phytanic acid had a different effect from a-tocopherol or phytol on temperature-dependent permeability of dipalmitoyllecithin liposomes (fig. 5). This difference can be explained by a difference in depth of the polar head groups of these phytyl compounds below the membrane surface. Cushley and Forrest [9] speculated that phytanic acid extends into the hydrophylic region and interferes with the normal ionic interaction between the negative phosphate of one lecithin molecule and the trimethylammonium group of adjacent lipid molecules, where ct.tocopheroi and phytol probably do not penetrate as far into the plane of the lecithin head group. In vitamin E deficiency, the activities of some phospholipid-activited membrane enzymes decrease [23,24], whereas those of others increase [ 2 5 - 2 9 ] . The functions of vitamin E may be affected by differences in the fatty acid species of the phospholipids associated with the enzyme activities.

K. Fukuzawa et al., Tocopherol and lecithin liposomes

21

Acknowledgements The authors wish to thank Dr. A. Suzuki for this helpful discussions, and Dr. S. Kijima for his kind supply ofa-tocopherol analogs.

References [1] [2] [3] [4] [5] [6] [7] [8] [91 [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25 ] [26] [27| [28 ] [29J

J.A. Lucy and J.T. Dingle, Nature 204 (1964) 156 K. Fukuzawa, K. Hayashi and A. Suzuki, Chem. Phys. Lipids 18 (1977) 39 D. Papahadjopoulos, M. Cowden and H. Kimelberg, Biochim. Biophys. Acta 330 (1973) 8 Y. Nozawa, Seikagaku (Japan) 47 (1975) 52 J.K. Raison and E.J. McMurchie, Biochim. Biophys. Aeta 363 (1974) 135 F.A. Hommes, D.J. Mastebroek-Helder and I. Molenaar, Nutr. Metabol. 19 (1975) 263 K. Fukuzawa, Vitamin (Japan) 50 (1976) 47 B. Maggio, A.T. Diplock and J.A. Lucy, Biochem. J. 161 (1977) 111 R.J. Cushley and BJ. Forrest, Can. J. Chem. 55 (1977) 220 D. Schmidt, H. Steffen and C. yon Planta, Biochim. Biophys. Acta 443 (1976) 1 M.C. Pangborn, J. Biol. Chem. 188 (1951) 471 W. Stoffel, F. Chu and E.H. Ahrens, Anal. Chem. 31 (1959) 307 A. Chalvardjian and E. Rudnicki, Anal. Biochem. 36 (1970) 225 R.A. Demel, K.R. Bruckdorfer and LL.M. van Deenen, Biochim. Biophys. Acta 255 (1972) 321 B.D. Ladbrook, R.M. Williams and D. Chapman, Biochim. Biophys. Acta 150 (1968) 333 K. Inoue, Biochim. Biophys. Acta 339 (1974) 390 K. Fukuzawa, Y. Suzuki and M. Uchiyama, Biochem. Pharmacol. 20 (1971) 279 R.A. Demel, L.L.M. van Deenen and B,A. Pethiea, Biochim. Biophys. Aeta 135 (1967) 11 R.A. Demel, S.C. Kinsky, C.B. Kinsky and LL.M. van Deenen Biochim. Biophys. Acta 150 (1968) 655 A.T. Diplock, J.A. Lucy, M. Verrinder and A. Zieleniewski, FEBS Letters 82 (1977) 341 I. Aibarracin, F.E. Lassaga and R. Caputto, J. Lipid Res. 15 (1974) 89 M.E. Leibowitz and M.C. Johnson, J. Lipid Res. 12 (1971) 662 K. Fukuzawa and M. Uchiyama, J. Nutr. Sei. Vitamin. 19 (1973) 433 A.H. Nathans and A.E. Kitabchi, Bioehim. Biophys. Acta 399 (1975) 244 R.N. Farias, T.F.R. Celis, A.L. Goldemberg and R.E. Trucco, Arch. Biochem. Biophys. 116 (1966) 34 M. Fedalesova, P.V. Sulakhe, J.C. Yates and N.S. Dhalla, Can. J. Physiol. Pharmacol. 49 (1971) 909 J. Schroeder, Biochim. Biophys. Acta, 343 (1974) 173 K. Kawai, M. Nakao, T. Nakao and G. Katsui, Am. J. Clin. Nutr. 27 (1974) 987 C.E. Hulstaert and I. Molenaar, Internat. J. Vit. Nutr. Res. 46 (1976) 262