Surface behaviour of long-chain lipolytic products (a 1-to-1 mixture of oleic acid and diolein) spread as monomolecular films in the presence of long-chain triglycerides

COLLOIDS AND SURFACES

. ~ ~ ELSEVIER

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Colloids and Surfaces B: Biointerfaces 6(1996) 173 180

Surface behaviour of long-chain lipolytic products (a 1-to- 1 mixture of oleic acid and diolein) spread as monomolecular films in the presence of long-chain triglycerides S. Labourdenne a.b M.G. Ivanova a,c, O. Brass a,b A. Cagna b R. Verger ~* ~' Lahoratoire de Lipolyse Enzymatique, UPR 9025 de I'IFRC 1 Biologie Structm'ale et Microbiolo~ie du C.N.R.S., 31 Chemin Joseph Aiguier. 13402 Marseille Cedex 22. Frwwe b lnte@wial Technology Concept SARL, Axone, 69930 Saint-Cl~ment-Les-Places. Fram~e Department o[ Physical Chemistry. James Bourchier I. 1126 S~)/ia, Bulgaria

Received 12 June 1995: accepted 11 October 1995

Abstract Surfitce pressure molecular area curves of pure and mixed monolayers of lipolytic products (a mixture of oleic acid and 1,2-dioleoyl-sn-glycerol (OA-DO) at a 1:1 molar ratio), oleic acid (OA) and purified soybean oil (TG) spread at the argon-water interface were obtained. The results reveal that OA-TG and {OA-DO) TG mixtures behave ideally when spread over a subphase at pH 8.0. We also determined the surface pressure molecular area curves of lipolytic products (OA DO) at the argon water interface in the absence or presence of an excess of triglycerides. In the presence of an excess o1" TG. the films of OA DO can be compressed up to surface pressures identical to the collapse pressure of the O A I ) O when present alone. Furthermore. the fact that at the collapse pressure, the area occupied by the O A D O mixture was the same in both the absence and presence of TG showed that under the experimental conditions used, negligible amounts of lipolytic products were dissolved in the oil and aqueous phases at both pH 5 and pH 8. The fraction of the surface occupied by O A - D O molecules varied from 80 to 100% at surface pressures ranging from 18 to 30 mN m 1 KevwoM~s: 1.2-Diolein: Interracial tension; Monomolecular films; Oil drop: Oleic acid: Soybean oil: Surface density

1. Introduction A m o n g the v a r i o u s techniques available for m e a s u r i n g ( p h o s p h o ) l i p a s e kinetics, m o n o m o l e c u lar fihn t e c h n o l o g y at the air w a t e r interface has been extensively d e v e l o p e d a n d used in o u r l a b o r a tory [ 1,2]. W i t h this technique, synthetic m e d i u m acyl chain glycerides a n d p h o s p h o l i p i d s are mostly used as substrates because they can form quite stable films, a n d their r e a c t i o n products, unlike * Corresponding author. 0927-7765:96 $15.00 ~: 1996 Elsevier Science B.V. All rights reserved SSDI I)927-7765(95)01251-6

those of the long-chain c o m p o u n d s , rapidly d e s o r b into the a q u e o u s s u b p h a s e [3,4]. We recently d e v e l o p e d a new m e t h o d , using a non-tensioactive agent such as g - c y c l o d e x t r i n present in the a q u e o u s s u b p h a s e to t r a p the single long-chain lipolytic p r o d u c t s generated u p o n the hydrolysis of m o n o m o l e c u l a r films of long-chain glycerides [ 5 ] or p h o s p h o l i p i d s [ 6 ] . A l o n g similar lines, N u r y et al. [ 7 ] a d a p t e d , for m o n i t o r i n g the lipase hydrolysis of n a t u r a l long-chain triacylglycerol, the o i l - d r o p technique, which is well k n o w n in the field of surface chemistry. The a c c u m u l a t i o n of insoluble

174

S. Labourdenne et al./Colloids Smfaces B." Biointer/~wes 6 (1996) 173-180

hydrolysis products at the surface of the oil drop is responsible for the decrease in the oil water interracial tension (?'o/w), which is correlated with changes with time in the oil-drop profile. Labourdenne et al. [8] have described some potential applications of the oil-drop tensiometer to lipolytic enzyme kinetics. To monitor the lipase kinetics on natural long-chain triacylglycerol, these workers used soybean oil as the substrate. Soybean oil contains mainly unsaturated long-chain fatty acids, which are released by enzyme action at the oil water interface and remain there transiently because of their amphipathic character. With this technique, it is possible to monitor lipase kinetics by measuring the decrease in )'o/w with time, or alternatively by maintaining a continuous and regulated increase with time in the oil-drop volume (and surface) keeping the interfacial tension at a fixed-end-point value. In this way, the surface density of lipolytic products can be kept constant [8]. In order to correlate the increase with time in the oil-drop surface (or alternatively the decrease in ,'o/w) with the number of molecules of lipolytic products generated per unit time and unit surface, we have to know the equation of state (interracial tension molecular area) of these lipolytic products present at the oil-drop surface. Since this is a technically difficult task, we decided to use the relatively simple continuous compression method, using the classical monomolecular film technique to accurately determine the surface density of the lipolytic products as well as the fraction of the surface occupied by these lipolytic products at a given surface pressure. On the one hand, we have to assume, however, as a first approximation, that the surface behaviour of these lipolytic products will be comparable both at the oil water interface and at the argon water interface in the presence of added purified soybean oil (TG). On the other hand, it is clear from the data published by Constantin et al. [9], Carri6re et al. [10] and Rogalska et al. [11] that diacylglycerols and free fatty acids are the only lipolytic products to be found initially with several lipases, up to 10% hydrolysis. A mixture of oleic acid and 1,2-dioleoylsn-glycerol (OA DO), at a 1:1 molar ratio, was therefore used, in view of the lipolytic products

formed during the initial steps of hydrolysis of a T G droplet, catalysed by a 1(3) specific lipase [ 11 ]. The aim of the present study was to describe the surface behaviour and to accurately measure the fraction of the surface occupied by a monomolecular film consisting of an O A - D O mixture spread at the argon-water interface, in the absence and presence of excess TG.

2. Materials and methods

2.1. Lipids Oleic acid (OA), purity 99%, and 1,2-diolein (DO), purity 97%, were from Sigma. T G is a soybean oil marketed by the Lesieur company, containing oleic acid (above 22%), linoleic acid (above 50%), linolenic acid (less than 10%) and less than 0.1% free fatty acid [12]. This soybean oil was purified by Transbiotec (Axone, 69930 Saint-Cldment-Les-Places, France) on a column of silicic acid (Merck) equilibrated in hexane ethyl ether. The purified lipids, T G (2 mgml-1), OA and D O ( 1 0 m g m l 1) were prepared in chloroform. The total concentration of the O A - D O mixture at a l : 1 molar ratio was 1 mg m l - t

2.2. Monolayer experiments Surface pressure-molecular area curves were measured at room temperature on an aqueous subphase of 10mM Tris-HC1 ( p H 8 ) , 0.15M NaC1, 21 mM CaC12, 1 mM EDTA as well as on a subphase of 10 mM acetate-HC1 (pH 5), 0.10 M NaC1, and 22 mM CaCI2, which are the usual buffers used for measuring pancreatic and gastric lipase activities, respectively. The buffers were filtered through a 0.2 lain millipore membrane. A rectangular Teflon trough measuring 24.8 x 14.8 c m 2, w a s rinsed with ethanol, tap water and double-distilled water before use. Measurements were performed with KSV 2200 barostat equipment (KSV Helsinki, Finland). The surface pressure was measured with a Wilhelmy plate attached to an electromicrobalance [3]. The film was compressed with a mobile barrier at a constant rate of

S. Labourdenne et al./('olh>ids" Sur/aces B." Biointe~Jhccs 6

3 0 m m m i n x ( 4 4 4 0 m m 2 min ~). The surface behaviour of a fatty acid in aqueous media at various pH is very complex 1-13 17]. Effectively, OA does not in fact form completely stable monomolecular fihns because the ionised form of OA is partly soluble in water• We have obtained several surface pressure molecular area curves at various compression speeds and we have observed that the surface pressure molecular area curves depend on the rate of compression• Above 30 mm m i n - ~, the surface pressure molecular area curves are identical. This rate of compression was selected in order to minimise the desorption of OA into the aqueous subphase. Lipid samples in chloroform solution, with a volume ranging from 22 to 40 ~al, were spread using a microsyringe and an interval of 10 rain was allowed to elapse for the chloroform to evaporate before the compression commenced. All the measurements were performed under an argon atmosphere to prevent lipid oxidation [5,18,19]. A stoichiometric mixture of OA D O was selected so that suitable lipolytic products would be generated during the initial steps o1" TG hydrolysis. We obtained surface pressure molecular area curves with the ternary mixture (OA DO) TG in which the stoichiometric mixture OA D O (lipolytic products) was assumed to constitute a single component. O A - D O is a suitable mixture for mimicking the surface behaviour of the lipolytic products formed during the initial steps of hydrolysis {up to 10% hydrolysis) of T G droplets.

3. Results and discussion 3.1. Surlace characterisation of monomolecular fihns q/pur(tied soybean oil (TG)

Surface pressure molecular area curves of T G films spread over an aqueous subphase at pH 5 and 8 are given in Fig. 1. At both pH values, the surface behaviour was comparable, except for a small difference between the values of the collapse pressures: 12 and 13raN m ~ at p H 5 and p H 8 , respectively. As a matter of fact, the collapse pressure of pure triolein was previously reported to be 12.3mN m ] at p H 8 [19] as well as p H 2 [5].

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\ Area/molecule of T(; I 4~2 ) Fig. 1. Surface pressure versus molecular area cttrvcs of purified soybean oil m o n o m o l e c u l a r lihns and pure triolein. "l'he subphase was in) Tris HCI bu(ti~r (pH S), l[ J) acelale IICI buffer ( p H 5 ) in Ihe case of purified soybean ~il and [?,) Tris HC1 buffer ( p H 8 ) , ( a k ) doubled-distilled water IICI [pH 2 ) m the case of pure triolein.

At the collapse pressure point. TG occupied approximately 118 ~2 per molecule, compared to approximately 100 ~2 per molecule in the case of pure triolein, both at pH 8 [19] and at pH 2 [5]. Since glycerides are uncharged molecules, it is to be expected that their surface behaviour will not be affected by the pH value of the aqueous subphase• The chemical nature of the fatty acids (chain length, number and position of the double bonds of the acyl chainsj will, however, influence the mean area occupied per triacylglycerol molecule. This may explain why higher values were obtained with soybean oil(118 ~2)containing high proportions of linoleic and linolenic acids, than with pure triolein (100 ,~21. 3.2. Smjiwe behaciour o[OA TG mixed monomolecular.li hns

Fig. 2 shows the surface pressure molecular area curves obtained with OA fihns spread over an aqueous subphase at pH 5 and pH 8, in the absence and presence of TG. Pure oleic acid fihns spread and compressed over a subphase at pH 8 or pH 5 gave identical surface pressure molecular area curves below 22 m N m ~ confirming a negligible aqueous solubilisation. The apparent collapse

176

S. Labourdenne et al./Colloid~ Surjaces B: Biointer[aces 6 (1996) 173 180

so-called "acid soap" [21,22]. Under these conditions, the "acid soap" is characterised by a hydrop h i l i ~ h y d r o p h o b i c balance which prevents it from desorbing into either of the two liquid phases (oil or water). Furthermore, the breaking point observed by previous investigators [15,23] in the surface pressure-molecular area curves of the fatty acid corresponded to a transition between twodimensional phases (liquid expanded-liquid condensed transition), since it was clearly distinct from the collapse point. These surface pressure molecular area curves might therefore not be true thermodynamic equilibrium isotherms. The surface pressure values were plotted against the area per molecule for the OA molecule, in order to show the retention of T G in the surface film at surface pressures greater than the collapse pressure of the pure TG. If no T G molecules were retained in the OA film, a clear-cut transition would occur at the collapse pressure of T G ( 1 3 r a N m 1).

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Area/molecule of OA (A 2) Fig. 2. (A) Surface pressure versus molecular area curves obtained with OA in the absence and presence of various proportions of TG. The lipids were initially spread from a chloroform solution on a subphase Tris HCI buffer (pH 8). The pressures were plotted against the area per OA molecule. +, OA; ,t, OA-TG (75:25); O, OA TG (50:50); ± , OA TG (10: 90). (B) As above, but the lipids were initially spread from a chloroform solution on a subphase acetate HCI buffer (pH 5).

point (breaking point) of OA is around 2 2 m N m - l at pH 8 (see Fig. 2A) a n d 3 5 m N m latpH5 (see Fig. 2B). This drastic different in the surface behaviour of OA at acidic and alkaline pH values is a consequence of its ionisation state. In terms of the hydrophilic-lipophilic balance (HLB) classification based on Davies' scale, oleic acid has the value 1 and potassium oleate the value 20 [20]. Oleic acid, therefore, seems to have the tendency to be soluble in oil, and oleate to be soluble in water. At a pH value of 8.0 (Fig. 2A), which is almost the apparent p K , of the oleic acid, the fatty acid molecules are partly ionised and form a

Fig. 3 shows the surface pressure-molecular area curves obtained with a stoichiometric O A - D O mixture spread over an aqueous subphase at pH 8 and 5, in the absence and presence of TG. To minimise the solubilisation of OA in the aqueous subphase, we adjusted the rate of compression to 30 m m rain 1 [13,17]. The surface pressure values were plotted against the mean area per molecule of the O A - D O mixture in order to show the retention of T G in the surface film at surface pressures higher than the collapse pressure of the pure TG. If no T G molecules were retained in the O A D O film, a clear-cut transition would occur at the collapse pressure of T G ( 13 m N m 1). When the surface pressure increased progressively above the collapse pressure of TG, however, the mean area per molecule of the pure O A - D O mixture became similar to the area of the O A - D O mixture, measured in the absence of TG. A similar behaviour was previously observed with a monomolecular mixed film of phosphatidylcholine and triacylglycerol [19,24]. In the presence of TG at surface pressures greater than 22 mN m 1, the

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area per molecule of mixed monomolecular fihns composed of OA TG (A, B} and (OA DO) TG IC, D) at surface pressures of 5raN m l (A, C) and 10mN m ~ (B. D}. The subphase was acetate HCI buffer qpH 5) or (at) "l"ris HCI buffer (pH8l, With mixtures of (OA DO} TG at pH 5, the points give the average of two separate ineasurelnents.

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Fig. 3. (A/ Surface pressure versus molecular area curves obtained with a stoichiomctric mixture of OA DO in the absence and presence of various proportions of TG. The lipids were initially spread from a chloroform solution on subphase Tris HCI buffer (pH 8) bet\~re the start of the compression. The sud:ace pressure wdues were plotted against the mean area per OA DO molecule. + OA DO; at,(OA DO TG(75:25): (OA DO) TG 150:50); O, (OA DO) TG (25:75): (OA DO) TG 110:90). (B) As above, but the lipids were initially spread from a chloroform solution on subphase acetate It(1 bufferlpH 51.

mean area per molecule of the O A - D O mixture was lower at pH 5.0 (see Fig. 3B) than at pH 8.0 (see Fig. 3At. 3.4. Comparison between the su@u'e behaviour of (OA DO) TG amt OA TG mixed monomolecular tilms Fig. 4 gives plots (derived from the data presented in Figs. 2 and 3) of the mean area per

molecule of mixed monomolecular films formed by (OA DO) TG and OA TG at two surface pressures (5 and 10mN m ~) and two pH values (5 and 8) of the aqueous subphase. The theoretical line connecting the two values corresponding to pure compounds indicates ideal mixing behaviour. Both the OA T G mixtures at pH 5 and 8 and (OA DO) T G at pH 8 actually behaved like ideal mixtures. Furthermore, the data obtained with {OA DO) TG mixtures at pH 5 deviated substantially from the ideal. A condensing effect occurred which was noticeable but relatively small, since it amounted to approximately only 8 9%, and did not vary significantly with the molar fraction of T G added to the OA DO mixture. 3.5. Sudace behaciour ql'the OA DO mixture in the absence or presem'e ql'an e\cess q[ TG We performed lipolysis of T G with pancreatic and gastric lipases using the pH-stat method (T.T.T. 60 Radiometer, Copenhagen, Denmark) at 37 C under non-optimised assav conditions (in

178

~K Lahourdenne e t aL / Colloids Su;jaces B." Biob~ter/iu'es 6 (1996) 173 180

the presence of 0.9% NaC1 alone). Lipolysis, up to 10% hydrolysis, was stopped by adding 10 ml of chloroform and the neutral lipids were also extracted with chloroform. After preparative T L C (2 mm silicagel Merck 60 F2s4), diglycerides and free fatty acids were recovered by performing a Folch's extraction procedure. These lipolytic products were then dissolved in chloroform and spread at the argon water interface in order to measure their surface pressure molecular area curves. In the absence of T G and at the same rate of compression, the surface behaviour of these complex natural lipolytic products were identical to the 1:1 OA D O mixture (data not shown). In order to simulate the surface behaviour of a stoichiometric mixture of OA D O in an oil drop undergoing lipolysis, we first spread at the argon water interface an amount of T G corresponding to 11 times the amount sufficient to form a true monomolecular film. We probably formed TG multilayers and small oil domains, in equilibrium with a collapsed T G monolayer at a value of 13 m N m ~. We then spread over the above T G layer a stoichiometric mixture of OA D O and then started the film compression procedure. Fig. 5

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Mean molecular area of the (OA-DO) mixture (~2) Fig. 5. Surface pressure versus molecular area curves obtained with a stoichiometric mixture of OA DO in the absence and presence of a collapsed T G phase (equivalent to 11 monolayers of TG). The lipids were initially spread from a chloroform solution on subphase Tris HCI buffer (pH 8) and acetate HCI buffer (pH 5) before the start of the compression. The surface pressure values were plotted against the mean area per OA D O molecule. :), no T G {pH 51; O, T G (pH 5); g], no TG tpH 81; II, T G (pH 81.

shows the surface pressure-molecular area curves of the OA D O mixture spread at the argon water interface in the absence and presence of an excess of TG. The two subphases used were either Tris HCI buffer (pH 8) or acetate HCI buffer (pH 5). It can be clearly seen from the figure that at all the surface pressures tested, the mean area per molecule of the OA DO mixture was smaller at pH 8 than at pH 5. This finding can be explained by the fact that the condensing effect was probably higher at pH 8.0 due to strong intermolecular hydrogen bonding between the ionised OA and D O molecules. It is worth noting that pure OA films gave identical surface pressure-molecular area curves at pH 8 or pH 5 (see Figs. 2A and 2B), which confirms that the solubility into the aqueous phase was negligible. Levy et al. [25] have shown that the interactions between various fatty acids and phospholipids, spread over a distilled water subphase, depend mostly upon the hydrophobic part and the presence of a double bond in the acyl chain. In the presence of TG, the mixed film of O A - D O can be compressed up to surface pressures identical to the collapse pressure of the OA DO mixture alone. Furthermore, the fact that at the collapse pressure, the area occupied by the OA DO mixture was the same in both the absence and the presence of T G showed that under the experimental conditions used, negligible amounts of lipolytic products were dissolved in the oil and aqueous phases at both pH 5 and pH 8. This important conclusion is in agreement with the data obtained previously by Small [21] and it is clear that the presence of a collapsed T G phase had no influence on the values of the area occupied by the OA D O mixture at either pH 8.0 or 5.0. The data in Fig. 5 could be used potentially with the monomolecular fihn technology, to estimate the enzymatic lipolysis rates of long-chain T G by maintaining a constant surface pressure at a given end-point value (above 13 m N m 1) by expanding the film area to compensate for the increase in the surface pressure [26]. With the data in Fig. 5, it is possible to calculate the total amount of lipolytic products produced per unit of time on the surface of a single oil-drop surface during lipolysis. Furthermore, when comparing the mean surface density of the OA D O

S. Lahourdenne el al./Colloids Surliwes B: Biointediwes 6 ( 1996j 173 180

179

Table I Surface density of OA DO at the argon buffer interface in the absence and presence of TG as a function of the surface pressure, and lhe percentage of the surface occupied by OA D O molecules Surface pressure (ran m '1

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Surface density of the O A - D O mixture (moleculecm :) x 10 1:~ -

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mixture in the absence and presence of TG, at a given subphase pH value, one can simply calculate the fraction of the surface which is occupied by lipolytic products and thus deduce the complementary fraction occupied by T G (Table 1). In our experiments, a fraction of the lipolytic products was probably solubilised into the oil phase due to the spreading technique used with mixed chloroform solutions. It is worth noting that the fraction of the surface occupied by O A - D O molecules ranges fl'om 80 to 100% at surface pressures ranging from 18 to 3 0 r a n m ~. On the other hand, the desorption rates of fatty acid are known to depend strongly on the chain length of the acyl chain. In fact, the rate constant of the desorption of laurie acid is approximately 2 × 10 4 s ~ at 11 mN m t [273 and that of myristicacid approximately 2 × 10 5 s 1 at 10 m N m ~ [28]. We studied the desorption rates of OA and mixed OA DO films, keeping a constant endpoint value of the surface pressure in the presence and absence of T G (data not shown). The decrease in the film area with time was monitored. With OA and OA DO, we obtained desorption rate constants around 10 s s - t which is comparable to the rate of desorption of myristic acid [28]. As was to be expected, when OA was tested alone at the argon water interface, the rate of desorption was higher at p H 8 than at p H 5 . At both pH values tested, the rates of desorption of OA and OA D O films were lowered, by a factor of around two, in the presence of TG (data not shown). It

20

N1 83 90 84 96 90 100 95

was therefore concluded that the T G multilayers retained the amphiphilic lipolytic products (OA and OA DO) at the interface. If one assumes that the specific activity of a lipase molecule on triolein is around 120 btmol min ~ mg 1 the corresponding catalytic rate constant (turnover number) in the case of a poorly active enzyme [11] will be 1 0 2s 1 It emerges quite clearly that the rate constant of product (OA DO mixed films) desorption {10 s s t) is negligible during the course of lipase action. The production of lipolytic products at the interface is therefore around 10~ times faster than their desorption. From the present data we established a correlation between the interracial tension and the surface density of the lipolytic products. It is subsequently proposed to estimate the lipase specific activity with the oil-drop tensiometer, once the interfacial excess of enzyme has been determined. Work is in now progress along these lines.

Acknowledgements This research was carried out in the framework of the B R I D G E T-Lipase Programme ( B I O T - C T 91-0274), the B I O T E C H G - P r o g r a m m e (BIO2-CT 94-3041) and the VALUE Programme (CTT 402) of the European Union. English revision by Dr. J. Blanc is acknowledged.

180

S. Labourdenne et al./Colloids Surfaces B." Biointer~lces 6 (1996) 173 180

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[13] 1. Jalal and G. Zografi, J. Colloid Interface Sci., 68 (1978) 196. [14] R.O. Scow, Biochimie, 70(1988) 1251. [15] B. Sims and G. Zografi, Chem. Phys. Lipids, 6 (1971 } 109. [163 J.F. Baret, H. Hasmonay, J.L. Firpo, J.J. Dupin and M. Dupeyrat, Chem. Phys. Lipids, 30 (19821 177. [ 17] D.P. Cistola, J.A. Hamilton, D. Jackson and D.M. Small, Biochemistry, 27 (1988) 1881. [18] R.O. Scow, P. Desnuelle and R. Verger, J. Biol. Chem., 245 (1979) 6456. [19] T.G. Redgrave, M.G. lvanova and R. Verger, Biochim. Biophys. Acta, 1211 (1994) 229. [20] J. Por6, Les Editions Techniques des Industries des Corps Gras. Emulsions, Microdmulsions, l~mulsions Multiples, Neuilly sur Seine, 1992, p. 37. [21] D.M. Small, Polyunsaturated Fatty Acids in Human Nutrition, in V. Bracco and R.J. Deckelbaum (Eds.), Nestld Nutrition Workshop Series, Vol. 28, 1992, p. 25. [22] G. Benzonana, Biochim. Biophys. Acta, 151 (19681 137. [23] A.A. Bois, 1.I. Panaiotov and J.F. Baret, Chem. Phys. Lipids, 34 (1984) 265. [24] G. Pidroni and R. Verger, J. Biol. Chem., 254 {1979) 10 090. [25] M.Y. Levy, S. Benita and A. Baszkin, Colloids Surfaces, 59 (19911 225. [26] J.W. Lagocki, N.D. Boyd, J.H. Law and V.J. Kezdy, J. Am. Chem. Soc., 92 (1970} 2923. [27] L. Ter Minassian-Saraga, J. Chem. Phys., 52 (1955) 181. [28] L. Ter Minassian-Saraga, C. R. Acad. Sci., 231 (1950) 337.