Physical Characterization and Lipase Susceptibility of Short Chain Lecithin/Triglyceride Mixed Micelles POTENTIAL LIPOPROTEIN MODELS*

(Received for publication, November 10, 1980)

Ramon A. Burns, Jr. and Mary Fedarko Roberts$ From the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Short chain lecithins form optically clear, stable mi- cellar particles with the triglycerides tributyrin and trihexanoin; they serve as effective substrates for pan- creatic lipase, cobra venom phospholipase Az, and phospholipase C from Bacillus cereus. These mixed lipid particles can contain as much as 0.5 mol fraction of triglyceride, depending on both lecithin and triglyc- eride fatty acyl chain length. Gel filtration studies of diheptanoyl phosphatidylcholine/trihexanoin (5:l) confirm that the triglyceride elutes with the lecithin and indicate a broader size distribution (with a slight decrease in apparent molecular weight) than pure lec- ithin micelles. Both components in these particles were studied by natural abundance 13C NMR spectroscopy. Lecithin motion and conformation is not detectably perturbed by the presence of triglyceride; 13C chemical shifts and spin-lattice relaxation times (TI) are unal- tered. Triglyceride chain motional behavior in the mixed particles mimics that of neat anhydrous triglyc- eride. In both instances, the chain TI profile is inter- mediate between that observed for monomeric triglyc- eride in CD30D (monotonic increase in TI down the alkyl chain) and for micellar lecithin (plateau region in the initial portion of the chain). Triglyceride backbone carbons show a pronounced motional anisotropy in the lecithin mixed particles that differs from the neat or monomeric triglyceride behavior. The effect of Mn2+ on the NMR peak intensities suggests sequestering of triglyceride from the aqueous phase.

Lipase hydrolysis rates of the triglyceride in these small, relatively homogeneous particles are 0.3-0.5 times those of triglyceride emulsions alone. Once suf- ficient lecithin is added to solublize the triglyceride, the reaction rate becomes independent of the lecithin/tri- glyceride ratio. This particle specific activity depends on both triglyceride and lecithin chain length. Phos- pholipase Az and C activities are not affected by up to 50 mol 5% triglyceride in the particle.

The NMR studies and enzymatic assays are discussed in terms of two models for the mixed particle: (i) the majority of the triglyceride is solubilized in a hydro- phobic core covered with a monolayer of lecithin and executes motions similar to neat triglyceride; and (ii) a fractional amount of triglyceride is at the surface with lecithin and in fast exchange with core lipid. Models

* This work was supported by National Institutes of Health Divi- sion of Research Resources Grant RR00995 and by National Science Foundation Contract C-670. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by National Institutes of Health Grant GM 26762 and National Science Foundation Grant PCM 7912622.

where the triglyceride is intermixed with lecithin at the micelle surface are seemingly excluded by the data.

The natural substrates of lipoprotein lipases and pancreatic lipases are mixed micellar lipid aggregates. Lipoproteins con- sist of a monolayer of phospholipid, cholesterol, and protein, surrounding a triglyceride/cholesterol ester core (1, 2). Bile lipid micelles contain a variety of fatty acids, glycerides, and phospholipids (3). Their physical properties are dependent on their composition and, although binary micelles of bile salts and phospholipids have been investigated (4,5), more complex mixtures or bile salt/triglyceride systems have not been ex- amined.

As a first step toward understanding the naturally occurring micellar structures, we have developed model mixed lipid particles using short chain lecithins as a micellar matrix. Short chain lecithins form micelles in aqueous solution ( 6 4 , where their detergent properties allow them to solubilize large frac- tions of lipophilic substances such as triglycerides. These relatively s m d , optically clear particles are easily and repro- ducibly formed, and the triglyceride is readily hydrolyzed by pancreatic lipase. They can be characterized with a variety of physical techniques. Short chain lecithins give rise to well resolved natura1 abundance 13C spectra in which aII carbons are observed and sn-l/sn-Z chemical shift differences are seen in micelles (9). By proper choice of phosphatidylcholine and triglyceride chain lengths one can observe resonances from all alkyl carbons and, hence, investigate motions with spin-lattice relaxation studies. This type of analysis leads to detailed models of lecithin/triglyceride micelles which can be corre- lated with lipase and phospholipase activity.

MATERIALS AND METHODS

Lipids-Dihexanoyl-PC’ (Calbiochem), tributyrin (Aldrich), and trihexanoin (Supelco and Sigma) were used without further purifica- tion. Triglyceride batches had less than 1.5% total impurities, 0.5% as free fatty acid, as determined by the ‘H NMR spectrum of the u-CH2 region where triglyceride can be separated from fatty acid, diglyceride, and monoglyceride.2 Diheptanoyl-PC and dioctanoyl-PC were syn- thesized by the fatty acid imidazolide method and purified by silicic acid chromatography (10). Phospholipid purity was checked by thin layer chromatography (9). Neat anhydrous triglyceride samples were prepared by evacuation a t low pressure in the presence of PZOS for 12 h. Mixed lecithin/triglyceride micelles were formed by co-solubiliza- tion of both lipids in benzene and/or chloroform, solvent removal under N2, and evacuation a t low pressure for a t least 2 h, addition of aqueous solution, and incubation for 4 h at room temperature. Tri-

’ The abbreviations used are: PC, phosphatidylcholine; diacyl-PC, 1,2-diacyl-sn-glycero-3-phosphorylcholine; TI, spin-lattice relaxation time; PIPES, 1,4-piperazinediethanesulfonic acid.

M. F. Roberts, unpublished results.

2716

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Short Chain Lecithin/Triglyceride Mixed Micelles 2717

glycerides formed optically clear solutions only when lecithin was present.

Enzymatic Assays-Phospholipase A2 (Nuju nuju nuja) and phos- pholipase C from Bacillus cereus were purified as described elsewhere (11, 12). Porcine pancreatic lipase I was obtained from Worthington. Enzymatic activity toward pure lecithin micelles, mixed micelles, and triglyceride emulsions was measured by pH-stat (13) a t 25°C. Assay mixtures contained 5 mM triglyceride, 0 to 55 mM lecithin, and 5 mM CaCI2. Lipid samples were co-solubilized in benzene and/or chloro- form directly in the assay vessels. Solvent was removed under N2 and then evacuated at low pressure for a t least 2 h. Distilled water then was added with thorough mixing, often by bath sonication for 10 s, and the samples incubated for 4 h a t room temperature.

Gel Filtration-Ascending gel fdtration chromatography utilized a column (56 X 2.5 cm) of Sepharose 6B equilibrated with 50 mM PIPES, I mM EDTA, pH 7.0, for standardization runs, and with 1.2 mM diheptanoyl-PC added for micelle sizing. The column was cali- brated by the optical elution of blue dextran (Sigma: M , = 2,000,000 used as a void volume marker), horse ferritin (Mann Research Labs, Inc.; M, = 341,000) and human hemoglobin (Sigma; M , = 64,000), and enzymatic assay of yeast alcohol dehydrogenase (14) (Worthington; M, = 141,000). Protein or lipid samples were placed in 1.5 ml of 50 mM potassium phosphate, 1 mM EDTA, pH 7.0, and applied to the column. Micelle samples contained 130 mM diheptanoyl-PC or 111 mM diheptanoyl-PC/22 mM trihexanoin. Phospholipid peaks and phosphate (column volume marker) were determined by phosphorus assay (15). Relative diheptanoyl-PC and trihexanoin content were determined by pooling the appropriate column fractions, lyophiliza- tion, extraction with CHClj:CHsOH (2:1), and analysis by ",IC NMR spectroscopy in CDaOD where both components are monomeric. Comparable backbone carbon peaks in both triglyceride and lecithin have similar nuclear Overhauser effects in CD.,OD (CHO, 1.8; CH10, 2.8). This allowed us to monitor triglyceride and lecithin relative concentrations in spectra without gated "H decoupling.

NMR Spectroscopy-""C NMR spectra were obtained a t 67.9 MHz with a Bruker 270 spectrometer equipped with a Nicolet 1080 data system. Spin-lattice relaxation times were measured as described previously (9). Samples contained 80 to 120 mM lecithin and 11 to 24 mM triglyceride in 50 mM potassium phosphate, 1 mM EDTA, in DrO, pD 7.4, a t 303 K. Samples were prepared as described under "Lipids;" however, 8-12 h of evacuation a t low pressure were used. Spectra of neat triglyceride were run in a 5-mm coaxial tube with DeO (lock frequency) in the exterior compartment.

Lipid samples for Mn2+ line-broadening experiments were prepared in 50 mM PIPES, 1 mM EDTA, 0.8 mM 2-['"C]acetate, pD 7.4. To circumvent problems of accurately measuring '"C line widths and/or accurately adding low concentrations of Mn" to the samples, we adopted the following analytical procedure. Mn"-induced line broad- ening was monitored by peak height changes in successive spectra taken under identical conditions. Selective Mn" broadening occurred with micromolar to tenths of millimolar metal ion in excess of EDTA. At higher Mn" concentrations (1-5.5 mM), broadening of the internal D20 lock signal caused all peaks to lose intensity at the same linear rate (7 (r+_3)% intensity lOSS/mM Mn"). Extrapolation of these lines for each carbon yielded an intensity, ZI, which is less than or equal to ZO (the experimental intensity in the absence of Mn2' or where the MnL+ is less than the EDTA concentration). By comparing II to Io we have generated a fractional intensity for each carbon, AI = (II - I(,)/ ZO, which reflects selective initial broadening due to Mn'+ proximity.

RESULTS

Mined Particle Formation-In aqueous solution, tributyrin and trihexanoin form emulsions which phase-separate if left unstirred. Trihexanoin appears cloudy in these suspensions. If the triglycerides are co-solubilized with short chain lecithins, optically clear solutions are obtained. Using optical clarity and absence of phase separation to monitor particle formation, we find that both dihexanoyl-PC and diheptanoyl-pc solubi- lize up to 0.25 mol fraction of tributyrin, while dioctanoyl-PC can form a homogeneous solution 1:l with this triglyceride. Diheptanoyl-PC can solubilize up to 0.3 mol fraction of tri- hexanoin. These particles are stable for several weeks at room temperature.

Fig. 1 shows the phosphate profiie for gel filtration of diheptanoyl-PC micelles and diheptanoyl-PC/trihexanoin (111 mM/22 mM) mixed micelles. Examination of the pooled

lipid peak in CDsOD by C'" NMR spectroscopy as described under "Materials and Methods," yields a diheptanoyl-PC/ trihexanoin ratio of 8.1 by integration of the CHO and CHZO peaks; the calculated ratio is 7.7 (this value includes the background of monomer lecithin present on the column). This indicates that essentially all the triglyceride elutes with the lecithin. As further confirmation, the fractions extending from the void volume to molecular weights slightly above the lipid peak were analyzed spectrally, and no trihexanoin was de- tected under conditions used to quantify the mixed lipid solution. There is a small decrease in apparent molecular weight for the mixed particle (93,000) relative to the dihep- tanoyl-PC micelle (102,000). In addition, the width at half- height for particle size distribution is approximately 1.5 times that of the pure lecithin. These molecular weights are only qualitative indicators of size since the column is calibrated with globular proteins, and light-scattering studies suggest short chain lecithins form nonspherical micelles (6 , 7). How- ever, the column size agrees moderately well with light-scat- tering values of 80,000-100,000 for diheptanoyl-PC at compa- rable concentrations (6, 7). Therefore, at concentrations nec- essary for '"C NMR studies, the diheptanoyl-PC/trihexanoin particle and diheptanoyl-PC micelle do not differ drastically in size.

I3C NMR Spectroscopy of Mixed Particles-High resolu- tion natural abundance '"C NMR spectra of the alkyl chain region of dihexanoyl-PC/tributyrin (4:l) and diheptanoyl-PC/ trihexanoin (4:l) mixed particles are shown in Fig, 2. Reso- nances for all triglyceride and lecithin alkyl chain carbons are clearly resolved. Assignments for the phospholipid and tri- glyceride carbons are based on Lindeman-Adams parameters for paraffin '"C chemical shifts, with the acyl chains treated as the appropriate methyl esters (16). The tributyrin carbons are resolved in all short chain lecithin particles, while trihex- anoin alkyl carbons are only distinct in diheptanoyl-PC and dioctanoyl-PC particles. Tributyrin alone emulsified in DrO at similar concentrations does not yield detectable resonances; phase-separated tributyrin is less dense than D20, rises to the top of the tube, and is not sampled by the receiver coil.

Lecithin chemical shifts are not affected by the incorpora- tion of triglyceride. Chemical shift differences between lecithin sn-1 and sn-2 chains are unaltered at the level of -+2 Hz. For

BO Fer ADH Hb P I

S O I SO 250 350 v o l u m e ( m e t

FIG. 1. Elution profiles for 130 m M diheptanoyl-PC (0) and 111 mM: 22 l l ~ ~ diheptanoyl-PC/tributyrin (0) on a column (56 x 2.5 cm) equilibrated with 50 nm PIPES, 1 m EDTA, 1.2 mM diheptanoyl-PC, pH 7.0. Elution positions of standard proteins are indicated. Phospholipid and column volume phosphate concentra- tions were determined by phosphorus assay. Marker proteins are blue dextran (BD) , ferritin (Fer), alcohol dehydrogenase (ADH), hemoglo- bin ( H b ) ; inorganic phosphate (P i ) marks the column volume.

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2718 Short Chain Lecithin/Triglyceride Mixed Micelles

triglyceride only the a-methylene carbon exhibits chain non- equivalence. The splitting (12 Hz) is the same for monomeric triglyceride in CD30D, neat triglyceride, and lecithin-solubi- lized triglyceride. This contrasts with the changes observed between monomer and micellar lecithin where the a carbon shift difference decreases (12 to 6 Hz) upon aggregation and several other carbons show enhanced chain-chain none- quivalence (9).

Figs. 3 and 4 show alkyl chain Tl profiles for the two mixed particle systems, neat triglyceride, and monomeric triglyceride in CD30D. The TI profde for dihexanoyl-PC (Fig. 3) or diheptanoyl-PC (Fig. 4) in mixed particles is similar to that for pure lecithin micelles (9). The "plateau" characteristic of

2 5 4 3 6 7

2 4 3

3'

L 4 '

A

L I I I I

DDm 30 20 IO

FIG. 2. "C NMR spectra of the alkyl chain region of mixed micelles of (A) dihexanoyl-PC/tributyrin (4:l) and (B) dihep- tanoyl-PC/trihexanoin (4:l). All of the alkyl carbons are resolved and identified; lecithin carbons are numbered; triglyceride carbons are indicated by primed numbers. The chemical shift scale is refer- enced to external tetramethylsilane.

51

4 / TI

(sed

3l 2

o ' h ; ; : A i ,

Carbon Number

FIG. 3. Alkyl chain "C spin-lattice relaxation times for mon- omeric tributyrin in CDsOD (A), neat anhydrous tributyrin (0), lecithin-solubilized tributyrin (a), and the dihexanoyl-PC micellar matrix (0) as a function of the carbon atom position of the alkyl chains. TI values for sn-1 (open symbols) and sn-2 (filled symbols) carbons are shown where the chains are magnetically nonequivalent and identified.

/

o L ~ ~ ~ A ~ 7 8 -1

Carbon Number

FIG. 4. Alkyl chain 13C TI values for monomeric trihexanoin in CDzOD (A), neat anhydrous trihexanoin (a), and micellar trihexanoin ((3) in diheptanoyl-PC (0) as a function of the carbon atom position of the alkyl chains. TI values for sn-1 (open symbols) and sn-2 (fitfed symbols) carbons are shown where the chains are magnetically nonequivalent and identified.

aggregated lecithin chains is not significantly affected by the presence of up to 0.25 mol fraction of triglyceride. The TI profiles for the triglyceride alkyl chain carbons are similar for neat triglyceride and lecithin-solubilized triglyceride, although the mixed particle TI values are slightly longer at each indi- vidual chain position. Both neat and particle triglyceride TI values are of the same magnitude as the chain Tl values for tristearin in the melt phase at 7OoC (17). However, only five distinct methylene carbon resonances are observed for tri- stearin with no individual information for carbon atoms in the center of the chain. Generally, the short chain triglycerides exhibit longer TI values than tristearin for carbon atoms near the carbonyl group and shorter 2'1 values near the terminal methyl group.

Table I gives Tl values for lecithin and triglyceride back- bone CHO and CHzO resonances in a variety of physical states. Triglyceride TI values in the mixed particle are similar in magnitude to those for neat triglyceride; however, the ratio T~(cHo,/T~(cH,o~ depends dramatically on the physical state of the triglyceride. This ratio is predicted to be 2.0 for a motion- ally isotropic system (18). Although a monomer of triglycer- ide or lecithin might be expected to undergo anisotropic motion, these compounds have experimental values for the backbone approaching the isotropic limit. For lecithin, TI(CHO)/TI(CH,O) = 1.6 (kO.1) independent of the molecuIar ag- gregation state (monomer in CD30D or micellar); for neat triglyceride or monomers in CD30D, the ratio is also around 1.6, consistent with the value of 1.4 (A0.2) found for tristearin in the melt phase (17). In contrast, T~(cHo,/T~(cH,o, is 0.7 (k0.2) for triglyceride solubilized by lecithin. Thus, a motion or relaxation mechanism is observed for triglyceride in the particle which is not found in monomeric or neat triglyceride.

Mn2+ Line Broadening of 13C NMR Resonances-The paramagnetic ion Mn2+, which will preferentially broaden res- onances of aqueous components or lipid carbons in the inter- facial region, can be used to probe how much, if any, triglyc- eride is at the particle surface. Fig. 5 shows the backbone, headgroup, and acyl chain portions of 13C NMR spectra of diheptanoyl-PC/trihexanoin in the absence and presence of 1.6 mM Mn2+. The lecithin methylene carbons adjacent to the phosphate moiety have been significantly broadened by the addition of the paramagnetic ion; other backbone and head- group resonances show lesser degress of broadening. In the acyl chain portion of the spectra, lecithin carbon atoms 2 and

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Short Chain Lecithin/Triglyceride Mixed Micelles 2719

TABLE I

- Mixed lipid system

DiCsPC/TB Monomer, CD30D Particle

TB, neat DiC7PC/TH

Monomer, CD30D Particle

TH. neat

13C TI values for glycerol carbons of lecithin and triglyceride in mixed micelles CHIO CHO

PC" TG PC TG S S

0.52 (0.02)b 1.27 (0.03) 0.79 (0.15)b 2.17 (0.02) 0.16 (0.02) 0.21 (0.01) 0.26 (0.04) 0.11 (0.04)

0.26 (0.01) 0.42 (0.05)

0.77 (0.04) 1.31 (0.08) 0.13 (0.002) 0.21 (0.04) 0.21 (0.01) 0.17 (0.02)

0.16 (0.01) 0.23 (0.03)

1.7 (0.1) 0.5 (0.2) 1.6 (0.3)

1.7 (0.2) 0.8 (0.3) 1.4 (0.3)

a Abbreviations: PC, lecithin, TG, triglyceride; DiCGPC, dihexanoyl-PC; DiCTPC, diheptanoyl-PC; TB, tributyrin; TH, trihexanoin. * Data from Ref. 9.

I! A

I I 50 40 35 30

ppm from inlcmol ~3CtH3]Acelote

1

2 5 4 7

2' , 4' 6'

* I I I 5 0 -5 -10 ppm from tnlernd 13CH3] Acelole

-15

FIG. 5. 13C NMR spectra of mixed micelles of diheptanoyl- PC/trihexanoin (4:l) in the absence (A) and presence (B) of 1.6 m~ Mn2+. All lecithin carbons are identified by letters or numbers as in Fig. 2; comparable triglyceride carbons are identified by primed captions or numbers.

3 have also been broadened. Comparable triglyceride carbon atoms are always much less affected by Mn2+.

A full analysis of the data as summarized under "Materials and Methods" yields more quantitative results (Table 11). Mn*+-induced changes in lecithin backbone, head group, and carbonyl intensities can be averaged to yield A I A V G = -0.41 (f0.10), i.e. an average of 41% of the peak intensity loss can be attributed to specific Mn2+ broadening. The MAVG is less for the lecithin acyl chains (-0.19 (+0.06)), a value statistically different from headgroup effects. All the trihexanoin peaks display even less sensitivity to low concentrations of MnZ+ (AZAVG = -0.05 (+O.lO)); this intensity loss is significantly smaller than that of PIPES buffer peaks (AIAvG = -0.19 (f0.15)). The difference in average intensity loss between lecithin headgroup/backbone and trihexanoin peaks has a 99% probability of being significant by the normal deviate test. Even the difference in intensity loss between lecithin acyl

TABLE I1 The effect of Mn2' on intensities of lecithin and triglyceride in

mixed micelles Carbon atom AI'

Diheptanoyl-PC N(CHd3 CHzN

-0.40 (0.02) -0.32 (0.05)

choline CH20P -0.49 (0.08) glycerol CH20P CHO

-0.59' -0.34 (0.20)

CHzO -0.40 (0.13) c=o -0.30 (0.06) c"2sn.z -0.17 (0.03)

*n-1 -0.18 (0.05) c-3,-2 -0.24 (0.13)

sn-1 -0.26 (0.04) c-4,.2 -0.27 (0.06)

sn-1 -0.17 (0.08) c-5 -0.09 (0.03)

-0.17 (0.02) C - 6 -0.14 (0.05) c-7 -0.18 (0.05)

CHO -0.12 (0.15) CHzO -0.13 (0.07) C = O -0.06' c-2en.2 - c-3 0.08 (0.10) c-4 0.04 (0.14) c-5 C"6

-0.01 (0.09) -0.22 (0.02)

PIPES Piperazine CH2N -0.10 (0.06) Ethylene CHzN -0.10 (0.06)

Trihexanoin

sn- I ,3 -0.01 (0.25)

CH2S03 -0.36 (0.04)

a AZ is calculated as described under "Materials and Methods";

Only two intensity points were obtained before the line broadened

-, this low intensity peak was excluded from AZAVG calculation.

standard deviations are given in parentheses.

into the base-line.

chains and trihexanoin peaks has a, 70% probability of signif- icance. The scatter in A I within each group does not seem to contain structural significance. The differences in AZ at this level may be limited by experiment uncertainty; alternatively, this may demonstrate the dynamic structure of these micelles (19). Simple static placement of lecithin and triglyceride mol- ecules in the mixed particle would predict little effect of the Mn2+ on middle and end alkyl chain carbons and a more pronounced drop in broadening efficiency in the backbone region and would not explain the observed data. PIPES buffer carbon resonances show the type of specificity one expects. The sulfonate methylene carbon, presumably nearer the site of Mn2+ interaction is broadened ( A I = -0.36) much more than its adjacent methylene (AI = -0.10).

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2720 Short Chain Lecithin/Triglyceride Mixed Micelles

Triglyceride Susceptibility to Pancreatic Lipase-The tri- glyceride in short chain lecithin/triglyceride micelles is acces- sible to pancreatic lipase hydrolysis. The lecithin component in the micelles is not hydrolyzed by the enzyme (Table 111). Specific activities observed depend upon incubation time and sample composition. Addition of water to dried mixed lipid fiims often produces a cloudy solution which clarifies upon standing 2-3 h at room temperature. If mixtures are assayed without the incubation period, a rate equivalent to the tribu- tyrin emulsion rate is observed. After incubation, a different particle-dependent activity is observed.

Fig. 6 shows enzyme activity toward tributyrin as a function of lecithin concentration for incubated mixtures of dihexanoyl- PC/tributyrin. At concentrations of lecithin sufficient to sol- ubilize and clarify the triglyceride, a fixed activity is observed. Below the lecithin/triglyceride solubilization ratio, a rate in- termediate between that of the tributyrin emulsion and the mixed micelle is obtained. Low concentrations of dihexanoyl-

TABLE I11 Pancreatic lipase activity against optically clear short chain

lecithin/triglyceride particles Sample F“ Specific activityh

DiC6PC/TBC 0.2 53 (1) DiC7PC/TB 0.2 70 (1) DiCsPC/TB 0.5 97 (9) DiCaPC/TB 97 (8) DiC7PC/TH 0.2 18 (1) DiCsPC/TH 0.3 10 (0.1) TB emulsion 210 (40)‘ TH emulsion 34 (13)‘ DiC7PC‘ <0.3

d -

F, mole fraction of triglyceride of Fist optically clear sample in this series.

All assays contain 5 mM triglyceride, 5 mM CaC12; activity units = micromoles min” mg”. Specific activity is reported with the standard deviation in parentheses. ‘ Abbreviations: D i c ~ P c , dihexanoyl-PC; DiC7PC, diheptanoyl-PC;

DiCsPC, dioctanoyl-PC; TB, tributyrin; TH, trihexanoin. -, includes two concentrations above lecithin concentration

which produces optical clarity. e Note reproducibility of mixed micelle assay relative to emulsion

assays. ’Lecithin concentration was 5 mM; no triglyceride present.

300 I

PC (1 mM) show a slight but statistically significant activation of lipase toward tributyrin. Lipase activity toward tributyrin as a function of diheptanoyl-PC and dioctanoyl-PC (Fig. 7 ) concentration does not show this lecithin monomer activation, but does show a fixed particle activity upon formation of optically clear micelles.

Lipase specific activity toward optically clear solutions and toward triglyceride emulsions is summarized in Table 111. For the mixed micelles, these data are averages for lecithin/tri- glyceride ratios above the solubilization point (shown for the dihexanoyl-PC/tributyrin system in Fig. 6). Chain length and substrate physical state trends are apparent. Lipase is approx- imately 5 times as active toward tributyrin emulsions as toward trihexanoin emulsions; it is 2.5 times as active toward the emulsions as toward the mixed lecithin/triglyceride mi- celles. With lecithin/tributyrin systems, enzyme activity in- creases as the host lecithin acyl chain length increases. The reverse occurs with lecithin/trihexanoin micelles.

Dioctanoyl-PC and tributyrin form stable, optically clear particles at equimolar concentrations (Fig. 7 ) . By itself at this concentration (5 mM) dioctanoyl-PC does not form optically clear solutions without the addition of KSCN or another salting-in compound. Additional lecithin in excess over the triglyceride solubilization limit causes the solution to become cloudy again. This suggests that excess lecithin phase-sepa- rates rather than dilutes the 1:l mixed micelle. Lipase activity is unaffected by additional lecithin (Table III; Fig. 7 ) .

!, cmc

200 i

0 20 4 0 Dioclonoyl - PC ( m M I

FIG. 7. Specific activity of porcine pancreatic lipase toward 5 mM tributyrin as a function of added dioctanoyl-PC concen- tration. cmc, critical micelle Concentration.

TABLE IV Phospholipase activities against short chain lecithin/triglyceride

particles Enzyme specific activity”

Sample Phospholipase Phosphoii- A., Dase C

DiC7PCh (20 mM) 500 (50) 250 (30) DiC7PC (20 mM)/TB (5 mM) 500 (50) 240 (10) DiC7PC (20 mM)/TH (5 mM) 370 (20) 260 (10) DiCsPC (5 mM) 3400 (400) 130 (10) DiCHPC (5 mM)/TB (5 mM) 3500 (800) 130 (20) TB (5 mM) t l (0.7

0 V 1 ” 2 0 60

Dlhesanoyl - PC I m M I

FIG. 6. Specific activity of porcine pancreatic lipase toward 5 mM tributyrin as a function of added dihexanoyl-PC concen- tration. cmc, critical micelle concentration.

“ All assays contain 5 mM CaCL; specific activity (units = micro- moles min” mg”) is reported with the standard deviation in paren- theses.

Abbreviations: DiC,PC, diheptanoyl-PC; DiCsPC, dioctanoyl-PC; TB, tributyrin; TH, trihexanoin.

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Short Chain Lecithin/Triglyceride Mixed Micelles 2721

Phospholipid Hydrolysis by Phospholipases-Table Iv shows the activity of cobra venom phospholipase A2 and B. cereus phospholipase C against several optically clear short chain lecithin/triglyceride mixed particles. Phospholipid mol- ecules with and without triglyceride are equally accessible to both phospholipases. Both these enzymes show kinetics which are very sensitive to small variations in phospholipid surface concentration (15,ZO). Only phospholipase A2 activity against the diheptanoyl-PC/trihexanoin system shows a slight rate decrease with added triglyceride. The dramatic phospholipase A2 activity increase for dioctanoyl-PC versus diheptanoyl-PC noted previously (21) is preserved in the presence of 5 mM tributyrin. Neither enzyme shows significant activity against a tributyrin emulsion alone (Table IV).

DISCUSSION

Structural Models for Short Chain Lecithin/Triglyceride Particles-Detailed mechanistic studies of the lipases and phospholipases have been hampered by poor understanding of surfaces and lack of experimental techniques applicable to the study of these interfaces. Manipulation of any variable in a surface active protein/biological interface system may alter enzymatic activity or physical properties in a myriad of ways. Careful studies of protein interaction with well characterized interfacial systems will continue to contribute in this field (22- 24). The short chain phosphatidylcholine micelles are partic- ularly useful in this respect. They possess a biologically rele- vant structure and conformation, yet form optically clear micellar solutions, making them favorable for conventional physical techniques (6-9, 24, 25). Their ability to solubilize triglycerides makes them particularly useful for lipase studies. In contrast, long chain phosphatidylcholines mixed with tri- glycerides tend to phase-separate, forming a phospholipid bilayer with a small amount of triglyceride incorporated, and triglyceride oil.

Several reasonable models for the short chain lecithin/short chain triglyceride particles can be proposed. The simplest model, that the triglyceride molecules are present among the lecithin molecules at the micelle interface, is inconsistent with (i) large differences between alkyl chain TI values for 2 mol- ecules forming a single mixed hydrophobic region, (ii) Mn2+ titration studies, and (iii) phospholipase activity. The Mn2+ effect on triglyceride carbons is significantly smaller than its effect on lecithin alkyl chain carbons or buffer. This suggests that most of the triglyceride does not reside at the particle surface. Both phospholipase A2 (20) and phospholipase C (15) show surface dilution behavior, i.e. hydrolysis depends on both bulk and surface phospholipid concentrations. The rate of hydrolysis is directly proportional to the mole fraction of lecithin (26). Extrapolation of the surface dilution shown by phospholipase hydrolysis of dipalmitoyl-PC/Triton X-100 mixed micelles (where the Triton X-100 has been shown to be a neutral surface dilutor (27)) predicts a rate decrease of about 50% for the 1:l dioctanoyl-PC/tributyrin mixed micelles. Both enzymes show unaltered activity in the presence of tributyrin. Comparison of diheptanoyl-PC/hexanol and diheptanoyl-PC/ trihexanoin (4:l) mixtures further suggests the triglyceride is not all a t the surface. When a diheptanoyl-PC/hexanol sample is prepared with a concentration of hexanol chains equivalent to trihexanoin fatty acyl chains (Le. 3 times the normal trihexanoin concentration), a stable, milky white emulsion is formed. Hexanol, present at the interface because of the hydrophilic character of the alcohol moiety, alters micelle packing so that a larger particle is formed.

In a second model for the particle, the triglyceride forms a hydrophobic core, which rigorously excludes short chain leci- thin mnlecules. The existence of a triglyceride core is sup-

ported by the similarity in TI profiles for alkyl chains in lecithin-solubilized triglyceride and for neat triglyceride. The lack of effect by triglyceride on lecithin parameters also is consistent. This mixed particle can be treated as a sphere for the sake of simplicity in calculation of its geometry. Using values for diheptanoyl-PC molecular length, volume, and sur- face area obtained elsewhere (7) and estimating similar param- eters for trihexanoin, we suggest a spherical particle contain- ing 16 trihexanoin molecules in a core covered with 77 phos- pholipid molecules. This particle has 0.17 mol fraction of triglyceride, 31 A radius, and M, = 53,000. For comparison, the phospholipid parameters generate a pure diheptanoyl-PC micelle which is a 150-molecule (Mr = 72,000) prolate ellipsoid with a = 225 A, b = 35 A. Given the crude approximations used in these calculations, the factor of two agreement with observed particle molecular weight and the amount of short chain lecithin required to solubilize trihexanoin is satisfactory.

Triglyceride in this system is not behaving motionally in quite the same fashion as neat triglyceride. The ratio T1(CHO)/TICH2O,, which is a measure of molecular anisotropy, is 1.6 for monomeric or neat triglyceride (the same ratio is found for all lecithin physical states) but is decreased to 0.7 for lecithin-solubilized triglyceride. Thus, the triglyceride does sense the presence of the lecithin. This may represent some alignment of triglyceride with the lecithin or a different con- formation from the tuning fork model of neat triglyceride (17).

Consistent with this surface/core model are the lipase ki- netic data showing that pancreatic lipase specific activity depends on fatty acyl chain length of lecithin as well as triglyceride. Pancreatic lipase alone does not bind to lecithin surfaces (28). If the enzyme interacts with core triglycerides, the lecithin chain length dependence should be small and the same for hydrolysis of tributyrin and trihexanoin. If triglyc- eride molecules are also present at the surface, then the lecithin activity dependence could reflect thermodynamic par- titioning of the triglyceride between the core and surface in each mixed particle. This will depend on alkyl chain lengths and on micelle sizes,

Lipase Hydrolysis of Micellar a n d Monolayer Triglycer- ide/Lecithin Systems-Detergents in general are very strong inhibitors of pancreatic lipase (28). Where they do not bind as micelles to the lipase, it has been suggested that the inhibition arises from the detergent interacting with the triglyceride emulsion interface. The structural view of the particles from NMR and enzyme studies places the triglyceride in a core with some small amount at the surface in fast exchange. Although further kinetics and particle characterization will be necessary to understand the observed dependence on both triglyceride and lecithin fatty acyl chain length in detail, we can compare these preliminary kinetics with other lipase/ triglyceride assay systems.

In a recent study of lipase activity toward didodecanoyl- PC/trioctanoin monolayers, an optimal enzyme specific activ- ity about twice that of the triglyceride alone was obtained around (0.7:l.O) lecithin/triglyceride (29). Surface (26) or “substrate” (29) dilution would predict that the specific activ- ity decreases with added lecithin. The monolayer kinetics for lecithin/triglyceride 5 1.0 then argue for an alteration in enzyme minimal specific activity (possibly an increase in kc,, or decrease in K,,,). For lecithin/triglyceride > 1.0, a decrease in enzyme specific activity was observed. Monolayer data is more difficult to obtain for these ratios since, as the number of triglycerides decreases, enzyme bound to the surface de- creases (i.e. the concentration of triglyceride is varying as well as the ratio lecithin/triglyceride). The activity decrease could be due to a further alteration in minimal specific activity or to a substrate dilution effect.

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2722 Short Chain Lecithin/ Triglyceride Mixed Micelles

Our mixed micelle system has lecithin/triglyceride ratios L 1.0. The action of pancreatic lipase on these short chain lecithin/triglyceride particles does not exhibit “surface dilu- tion” kinetics. As the lecithin/triglyceride ratio increases from the solubilization point, lipase specific activity remains con- stant for all particles up to a ratio of 81. The kinetics then indicate that either (i) total triglyceride and not surface con- centration of triglyceride is important in the rate behavior or (ii) the surface triglyceride (the actual lipase substrate) in a given lecithin/triglyceride system is unaltered with additional lecithin. The first suggests that core triglyceride is the site of lipase hydrolysis or that, if surface triglyceride is the prefer- ential substrate, surface binding of the enzyme and not catal- ysis is rate-limiting. The second would be the case if a ther- modynamically stable mixed particle with a unique composi- tion is formed, and excess lecithin segregates into pure phos- pholipid micelles. The actual particle specific activity, which depends on the acyl chain length of both components, could then reflect variations in amount of surface triglyceride avail- able to lipase. Such mixing behavior is atypical of most mixed micelles containing lipid (30) but cannot be eliminated at present for the short chain lecithin/triglyceride particles. In fact, the limited concentration range of particle clarity for dioctanoyl-PC/tributyrin suggests that a unique composition range may exist for that binary mixture. Experiments are in progress to characterize further our mixed lipid particles.

Model “MiniZipoproteins”-Mixed lipid particles using short chain lecithins as the micellar matrix can be exploited further as a step toward producing model lipoproteins. Up to 17 mol 5% cholesterol can be micellized by short chain leci- thins.” By next adding cholesterol esters one can form micelle particles with several compositional degrees of freedom. Ef- fects of added components on lecithin and triglyceride behav- ior can be characterized by ”’C NMR and lipase/phospholi- pase susceptibility; the entire particles can be probed by quasielastic light-scattering and Raman scattering. In essence, we can form a “minilipoprotein” without the protein mole- cules. The latter, or synthetic fragments, can then be selec- tively introduced and the biological consequences measured in a variety of systems. By understanding the detailed struc- ture and various lipid interactions in these model micellar particles we may better understand lipolysis and other enzy- matic modifications of lipoproteins or other biologically com- plex micellar particles.

Achnoulledgments-We would like to thank Ms. Maha El-Sayed for purification of phospholipase C from B. cereus. The NMR exper- iments were oerformed at the NMR facilitv for Biomolecular Re-

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