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 Departmentof Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts02139

Short chain lecithinsform optically clear, stable mi- where the triglycerideis intermixed with lecithin at the
cellar particles with the triglycerides tributyrin and micelle surface are seemingly excluded by the data.
trihexanoin; they serveas effective substrates forpan-
creaticlipase,cobra venom phospholipase Az, and
phospholipase C from Bacillus cereus. These mixed
lipid particles can contain as much as 0.5 mol fraction The natural substrates of lipoprotein lipases and pancreatic
of triglyceride, depending on both lecithin and triglyc- lipases are mixed micellar lipid aggregates. Lipoproteins con-
eride fatty acyl chain length. Gel filtration studies of sist of a monolayer of phospholipid, cholesterol, and protein,
diheptanoyl phosphatidylcholine/trihexanoin (5:l) surrounding a triglyceride/cholesterol ester core (1, 2). Bile
confirm that the triglyceride elutes with the lecithin lipid micelles contain a variety of fatty acids, glycerides, and
and indicate a broader size distribution (with a slight phospholipids (3). Their physical properties are dependent on
decrease in apparent molecular weight) than pure lec- their composition and, although binary micelles of bile salts

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ithin micelles. Both components in these particles wereand phospholipids have beeninvestigated (4,5),more complex
studied by natural abundance 13C NMR spectroscopy. mixtures or bile salt/triglyceride systems have not been ex-
Lecithin motion and conformation is not detectably amined.
perturbed by the presence of triglyceride; 13Cchemical As a first step toward understanding the naturally occurring
shifts and spin-lattice relaxation times ( T I )are unal- micellar structures, we have developed model mixed lipid
tered.Triglyceridechainmotionalbehavior inthe particles using short chainlecithins as a micellar matrix. Short
mixed particles mimics that of neat anhydroustriglyc- chain lecithinsform micelles in aqueoussolution ( 6 4 , where
eride. In both instances, the chain T I profile is inter- their detergent properties allow them to solubilize large frac-
mediate between that observed for monomeric triglyc- tions of lipophilic substancessuchas triglycerides. These
eride in CD30D (monotonic increase in TI down the relatively s m d , optically clear particles are easily and repro-
alkyl chain) and for micellar lecithin (plateau region in ducibly formed, and thetriglyceride is readily hydrolyzed by
the initial portionof the chain). Triglyceride backbone
pancreatic lipase. They canbe characterized with a variety of
carbons show a pronounced motional anisotropy in the
lecithin mixed particles that differs from the neat or physical techniques. Short chain lecithins give rise to well
monomeric triglyceride behavior. Theeffect of Mn2+on resolved natura1 abundance 13Cspectra in which aII carbons
the NMR peakintensitiessuggestssequestering of are observed and sn-l/sn-Z chemical shift differencesare seen
triglyceride from the aqueousphase. in micelles (9). By proper choice of phosphatidylcholine and
Lipase hydrolysis rates of the triglyceride in these triglyceride chain lengths one can observe resonances from all
small, relatively homogeneous particlesare 0.3-0.5 alkyl carbons and, hence, investigate motions withspin-lattice
times those of triglyceride emulsions alone. Once suf- relaxation studies.Thistype of analysis leadsto detailed
ficient lecithin is added to solublize the triglyceride, the models of lecithin/triglyceride micelles which can be corre-
reaction rate becomes independent of the lecithin/tri- lated with lipase and phospholipase activity.
glyceride ratio. This particle specific activity depends
on both triglyceride and lecithin chain length. Phos- MATERIALS AND METHODS
pholipase Az and C activities are not affected by up to Lipids-Dihexanoyl-PC’ (Calbiochem),tributyrin(Aldrich),and
50 mol 5% triglyceride in the particle. trihexanoin (Supelco and Sigma)were used without further purifica-
The NMR studies and enzymatic assays are discussed tion. Triglyceride batches had less than 1.5% total impurities, 0.5% as
in terms of two models for the mixed particle: (i) the free fatty acid, as determined by the ‘H NMR spectrum of the u-CH2
majority of the triglyceride is solubilized in a hydro- region where triglyceride can be separated from fatty acid, diglyceride,
phobic core covered with a monolayer of lecithin and and monoglyceride.2 Diheptanoyl-PC and dioctanoyl-PC were syn-
thesized by the fatty acid imidazolide method and purified by silicic
executes motions similarto neattriglyceride; and (ii) a acid chromatography (10).Phospholipid purity was checked by thin
fractional amount of triglyceride is at the surface withlayer chromatography (9). Neat anhydroustriglyceride samples were
lecithin and in fast exchange with core lipid. Models prepared by evacuation a t low pressure in the presence of PZOSfor 12
h. Mixed lecithin/triglyceride micelles were formed by co-solubiliza-
tion of both lipids in benzene and/or chloroform, solvent removal
* This work was supported by National Institutes of Health Divi- under N2, and evacuation a t low pressure for a t least 2 h, addition of
sion of Research Resources Grant RR00995 and by National Science aqueous solution, and incubation for 4 h at room temperature. Tri-
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 ’The abbreviations used are: PC, phosphatidylcholine; diacyl-PC,
with 18 U.S.C. Section 1734 solely to indicate thisfact. TI,
1,2-diacyl-sn-glycero-3-phosphorylcholine; spin-lattice relaxation
$ Supported by National Institutesof Health Grant GM 26762 and time; PIPES, 1,4-piperazinediethanesulfonicacid.
National Science Foundation Grant PCM 7912622. M. F. Roberts, unpublishedresults.

2716
 Lecithin/Triglyceride
Chain
ShortMicelles
Mixed
glycerides formed opticallyclear solutions only when lecithinwas
present.
Enzymatic Assays-Phospholipase A2 (Nuju nuju nuja) andphos-
pholipase Cfrom Bacillus cereus were purifiedas 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 incubatedfor 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 (MannResearch 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 peaksand
phosphate (column volume marker) were determined by phosphorus
assay (15). Relativediheptanoyl-PCand trihexanoin content were
determined by pooling the appropriate column fractions, lyophiliza-
tion, extraction with CHClj:CHsOH (2:1), and analysis by ",IC NMR
2717
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 valueincludes the
background of monomer lecithin present on the column). This
indicates that essentially all the triglyceride elutes with the
lecithin. As further confirmation, the fractionsextending from
the void volume to molecular weights slightly abovethe lipid
peak were analyzed spectrally, and no trihexanoin was de-
tectedunder conditions used toquantifythe mixed lipid
solution. There is asmall 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
withglobularproteins, and light-scattering studies suggest
short chain lecithins formnonspherical micelles ( 6 , 7). How-
ever, the column size agrees moderately well with light-scat-
tering valuesof 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-

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spectroscopy in CDaOD where bothcomponentsare monomeric. tion natural abundance '"C NMR spectra of the alkyl chain
Comparable backbone carbon peaks in bothtriglyceride and lecithin region of dihexanoyl-PC/tributyrin (4:l)and diheptanoyl-PC/
have similar nuclear Overhauser effects in CD.,OD (CHO, 1.8; CH10,
2.8). This allowed us to monitor triglyceride and lecithin relative trihexanoin (4:l)mixed particles are shown in Fig, 2. Reso-
concentrations in spectra without gated" H decoupling. nances for all triglyceride and lecithin alkyl chain carbons are
NMR Spectroscopy-""C NMR spectra were obtained a t 67.9 MHz clearly resolved. Assignments for the phospholipid and tri-
with a Bruker 270 spectrometer equipped with a Nicolet 1080 data glyceride carbons arebased on Lindeman-Adams parameters
system.Spin-lattice relaxation times were measuredas described for paraffin '"C chemical shifts, with the acyl chains treated
previously (9). Samples contained 80 to 120 mM lecithin and 11 to 24 as the appropriate methyl esters (16). The tributyrin carbons
mM triglyceride in 50 mM potassium phosphate, 1 mM EDTA, in DrO,
pD 7.4, a t 303 K. Samples were prepared asdescribed under "Lipids;" are resolved in all short chain lecithin particles, while trihex-
however, 8-12 h of evacuation a t low pressure were used. Spectra of anoin alkyl carbons are only distinct in diheptanoyl-PC and
neat triglyceride were run in a 5-mm coaxial tube with DeO (lock dioctanoyl-PC particles. Tributyrin alone emulsified in DrO
frequency) in the exterior compartment. at similar concentrations does not yield detectable resonances;
Lipid samples for Mn2+line-broadening experiments were prepared phase-separated tributyrin is less dense than D20,rises to the
in 50 mM PIPES, 1 mM EDTA, 0.8 mM 2-['"C]acetate, pD 7.4. T o top of the tube, andis not sampled by the receiver coil.
circumvent problems of accurately measuring '"C line widths and/or
accurately adding low concentrations ofMn" to the samples, we Lecithin chemical shifts are not affected by the incorpora-
adopted thefollowing analytical procedure. Mn"-induced line broad- tion of triglyceride. Chemical shift differences between lecithin
ening was monitored by peak height changes in successive spectra sn-1 and sn-2 chains are unaltered at the level of -+2 Hz. For
taken under identical conditions. Selective Mn" broadening occurred
BO Fer ADH Hb PI
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 ParticleFormation-In aqueous solution, tributyrin
and trihexanoin form emulsions which phase-separate if left
unstirred. Trihexanoin appearscloudy inthese suspensions. If
the triglycerides are co-solubilized with short chainlecithins,
optically clear solutions are obtained. Using optical clarity
and absenceof phase separation to monitor particle formation,
we find that both dihexanoyl-PC and diheptanoyl-pc solubi- SO I SO 250 350
lize up to 0.25 mol fraction of tributyrin, while dioctanoyl-PC volume ( m e t
can form a homogeneous solution 1:l with this triglyceride. FIG. 1. Elution profiles for 130 m M diheptanoyl-PC (0)and
Diheptanoyl-PC can solubilize up to 0.3 mol fraction of tri- 111 mM: 22 l l diheptanoyl-PC/tributyrin
~ ~ (0)on a column (56
hexanoin. These particles are stable for several weeks at room x 2.5 cm) equilibratedwith 50 nm PIPES, 1 m EDTA, 1.2 mM
temperature. diheptanoyl-PC, pH7.0. Elution positions of standard proteins are
indicated.Phospholipid and columnvolume phosphate concentra-
Fig. 1 shows thephosphate profiie for gel filtration of tions were determined by phosphorus assay. Marker proteins areblue
diheptanoyl-PC micelles and diheptanoyl-PC/trihexanoin dextran ( B D ) ,ferritin (Fer),alcohol dehydrogenase (ADH),hemoglo-
(111 mM/22 mM) mixed micelles. Examination of the pooled bin ( H b ) ;inorganic phosphate ( P i )marks the column volume.
2718 Lecithin/Triglyceride
Chain
ShortMicelles
Mixed
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 forthe 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
-1
o L ~ ~ ~ A ~
CarbonNumber
2 5 4 3 6 7
FIG. 4. Alkyl chain 13CTI values formonomeric trihexanoin
in CDzOD (A), neat anhydrous trihexanoin (a), and micellar
trihexanoin ((3) in diheptanoyl-PC (0) as a function of the
carbon atomposition of the alkyl chains. TIvalues for sn-1 (open
symbols) and sn-2 (fitfed symbols) carbons are shown where the
chains aremagnetically nonequivalent and identified.

aggregated lecithin chains is not significantly affected by the

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presence of up to 0.25 mol fraction of triglyceride. The TI
profiles for the triglyceride alkyl chain carbons are similar for
2 4 3 neat triglyceride and lecithin-solubilizedtriglyceride, although
3' 4' the mixed particle T I values are slightly longer at each indi-
A vidual chain position. Both neat and particle triglyceride T I
values are of the same magnitude as the chain Tl values for

I
30
I

DDm
LL I
20
I
IO
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
FIG. 2. "C NMR spectra of the alkyl chain region of mixed the carbonyl group and shorter 2'1 values near the terminal
micelles of (A) dihexanoyl-PC/tributyrin(4:l) and (B) dihep- methyl group.
tanoyl-PC/trihexanoin (4:l).All of the alkyl carbons are resolved Table I gives Tl values for lecithin and triglyceride back-
and identified; lecithin carbons are numbered; triglyceride carbons boneCHO and CHzO resonances in a variety of physical
are indicated by primed numbers. The chemical shift scale is refer- states. Triglyceride T Ivalues in the mixed particle are similar
enced to external tetramethylsilane.
in magnitude to those for neat triglyceride; however, the ratio
T ~ ( c H o ,/ T ~( depends
c H ,o ~ dramatically on the physical state of
the triglyceride. This ratiois 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-
4 / gregation state (monomer in CD30D or micellar); for neat
TI triglyceride or monomers in CD30D, the ratio is also around
(sed 51 1.6, consistent with the value of 1.4 (A0.2) found fortristearin

3l
2
in the melt phase (17). In contrast, T~(cHo,/T~(cH,o,

or relaxation mechanism is observed for triglyceride in the

is 0.7
(k0.2) for triglyceride solubilized by lecithin. Thus, a motion

particle which is not found in monomeric or neat triglyceride.

, facial region, can be used to probe how much, if any, triglyc-
o ' h ; ; : A
CarbonNumber
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 functionof the carbon atom position
of the alkyl chains. TIvalues for sn-1 (open symbols) and sn-2
(filled symbols) carbons are shown where the chains are magnetically
nonequivalent and identified.
Mn2+ Line Broadening of 13C NMR Resonances-The
paramagnetic ion Mn2+,which willpreferentially broaden res-
onances of aqueous components or lipid carbons in the inter-
i eride is at the particle surface. Fig. 5 shows the backbone,
headgroup, and acyl chain portions of13C 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
 Lecithin/Triglyceride
Chain
ShortMicelles
Mixed 2719
TABLE
I
- 13C TIvalues for glycerol carbons of lecithin and triglyceride in mixed micelles
CHIO CHO
Mixed lipid system
PC" TG PC TG
S S
DiCsPC/TB
Monomer, CD30D 0.521.27
(0.03) (0.02)b 0.79 (0.15)b (0.02)2.17 1.7 (0.1)
Particle (0.02)0.16 0.21 (0.01) 0.26 (0.04) 0.11 (0.04) 0.5 (0.2)
TB, neat 0.26 (0.01) 0.42 (0.05) 1.6 (0.3)
DiC7PC/TH
Monomer, CD30D 0.77 (0.04) 1.31 (0.08) 1.7 (0.2)
Particle (0.002)
0.13 0.21 (0.04) 0.21 (0.01) (0.02)0.17 0.8 (0.3)
TH. neat (0.01) 0.16 0.23 (0.03) 1.4 (0.3)
a Abbreviations: PC, lecithin, TG, triglyceride; DiCGPC, dihexanoyl-PC; DiCTPC, diheptanoyl-PC; TB, tributyrin; TH, trihexanoin.
* Data from Ref. 9.
TABLEI1
The effectof Mn2' on intensities of lecithin and triglyceride in

I! A Carbon atom
Diheptanoyl-PC
mixed micelles
AI'

N(CHd3 -0.40 (0.02)

CHzN -0.32 (0.05)
choline CH20P (0.08)
-0.49
glycerol CH20P -0.59'
CHO -0.34 (0.20)

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CHzO -0.40 (0.13)
c=o -0.30 (0.06)
c"2sn.z -0.17 (0.03)
*n-1 -0.18 (0.05)
I I 1 c-3,-2 -0.24 (0.13)
50 40 35 30 sn-1 -0.26 (0.04)
ppm from inlcmol ~3CtH3]Acelote
c-4,.2 -0.27 (0.06)
sn-1 -0.17 (0.08)
2 5 4 7
c-5 -0.09 (0.03)
2' , 4' 6'
-0.17 (0.02)
C-6 (0.05) -0.14
c-7 -0.18 (0.05)
Trihexanoin
CHO -0.12 (0.15)
CHzO (0.07) -0.13
C=O -0.06'
c-2en.2 -
sn- I ,3 -0.01 (0.25)
c-3 (0.10) 0.08
c-4 0.04 (0.14)

* 5
I
0
I
-5
ppm from tnlernd 13CH3] Acelole
I
-10 -15
c-5
C"6
PIPES
Piperazine CH2N
-0.01 (0.09)
-0.22 (0.02)

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 assummarized under "Materials
and Methods" yields more quantitative results (Table 11).
Mn*+-inducedchanges 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 issignificantly
smaller than that of PIPES buffer peaks ( A I A v G = -0.19
(f0.15)). The difference in average intensity lossbetween
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
-0.10 (0.06)
Ethylene CHzN -0.10 (0.06)
CH2S03 -0.36 (0.04)
a AZ is calculated as described under "Materials and Methods";
standard deviations are given in parentheses.
Only two intensity points were obtained before the line broadened
into the base-line.
-, this low intensity peak was excluded from AZAVGcalculation.
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).
2720 Lecithin/Triglyceride
Chain
Short Micelles
Mixed
Triglyceride Susceptibility toPancreatic Lipase-The tri-
glyceride in shortchain 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 timeand
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-
TABLEI11
Pancreatic lipase activity against optically clear short chain
lecithin/triglyceride particles
Sample F“ Specific activityh
DiC6PC/TBC 0.2
DiC7PC/TB 0.2
PC (1mM) 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
53 (1) triglyceride solubilization limit causes the solution to become
70 (1) cloudy again. This suggests that excess lecithin phase-sepa-

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DiCsPC/TB 97 0.5 (9) rates rather than dilutes the 1:l mixed micelle.Lipase activity
DiCaPC/TB -d 97 (8)
DiC7PC/TH 0.2 18 (1) is unaffected by additional lecithin (Table III; Fig. 7 ) .
DiCsPC/TH10 0.3 (0.1)
T B emulsion 210 (40)‘
T H emulsion 34 (13)‘ !, cmc
DiC7PC‘ <0.3
F, mole fraction of triglyceride of Fist optically clear sample in
this series.
200 i
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 cdihexanoyl-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

0 20 40
Dioclonoyl - PC ( mM I
FIG. 7. Specific activity of porcine pancreatic lipase toward
5 mM tributyrin asa 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)
(10)
260
DiCsPC (5 mM) 3400 (400) 130 (10)
DiCHPC (5 mM)/TB (5 mM) 3500 (800) 130 (20)
TB (5 mM) tl (0.7
0 V 20 1 ” 60
“ All assays contain 5 mM CaCL; specific activity (units = micro-
D l h e s a n o y l - PC I m M I moles min” mg”) is reported with the standard deviation in paren-
FIG. 6. Specific activity of porcine pancreatic lipase toward theses.
5 mM tributyrin asa function of added dihexanoyl-PC concen- Abbreviations: DiC,PC, diheptanoyl-PC; DiCsPC, dioctanoyl-PC;
tration. cmc, critical micelle concentration. TB, tributyrin; TH, trihexanoin.
 Short Chain Lecithin/Triglyceride Mixed Micelles 2721
PhospholipidHydrolysis by Phospholipases-Table Iv ported by the similarityin TI profiles for alkyl chains in
shows the activity of cobra venom phospholipase A2 and B. lecithin-solubilized triglyceride and for neat triglyceride. The
cereus phospholipase C against several optically clear short lack of effect by triglyceride on lecithin parameters also is
chain lecithin/triglyceride mixed particles. Phospholipid mol- consistent. Thismixed particle can be treated as a sphere for
ecules with and without triglyceride are equally accessible to the sake of simplicity in calculation of its geometry. Using
both phospholipases. Both theseenzymes show kinetics which values for diheptanoyl-PC molecular length, volume, and sur-
are very sensitive to small variations inphospholipid surface face area obtainedelsewhere (7) and estimating similar param-
concentration (15,ZO).Only phospholipase A2 activity against eters for trihexanoin, we suggest a spherical particle contain-
the diheptanoyl-PC/trihexanoin system shows a slight rate ing 16 trihexanoin molecules in a core covered with 77 phos-
decrease with added triglyceride. The dramaticphospholipase pholipid molecules. This particle has 0.17 mol fraction of
A2 activity increase for dioctanoyl-PC versus diheptanoyl-PC triglyceride, 31 A radius, and M , = 53,000. For comparison,
noted previously (21) is preserved in the presence of 5 mM the phospholipid parameters generatea pure diheptanoyl-PC
tributyrin. Neither enzyme shows significant activity against micelle which is a 150-molecule ( M r= 72,000) prolate ellipsoid
a tributyrin emulsion alone (Table IV). with a = 225 A, b = 35 A. Given the crude approximations
used in these calculations, the factor of two agreement with
DISCUSSION
observed particle molecular weight and the amount of short
Structural Modelsfor Short Chain Lecithin/Triglyceride chain lecithin required to solubilize trihexanoin is satisfactory.
Particles-Detailed mechanistic studies of the lipases and Triglyceride in this system is not behaving motionally in
phospholipases have been hampered by poor understanding quitethesame fashion as neat triglyceride. Theratio
of surfaces andlack of experimental techniquesapplicable to T 1 ( C H O ) / T I C H 2 O , , which is a measure of molecular anisotropy,
the studyof these interfaces. Manipulation of any variable in is 1.6 for monomeric or neat triglyceride (the same ratio is
a surface activeprotein/biological interface system may alter found for all lecithin physical states) but is decreased to 0.7
enzymatic activity orphysical properties in a myriad of ways. for lecithin-solubilized triglyceride.Thus, thetriglyceride does
Careful studies of protein interaction withwell characterized sense the presence of the lecithin. This may represent some
interfacial systems will continue to contribute in thisfield (22- alignment of triglyceride with the lecithin or a different con-

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24). The short chain phosphatidylcholinemicelles are partic- formation from the tuningfork model of neat triglyceride (17).
ularly useful in this respect. They possess a biologically rele- Consistent with this surface/core model are the lipase ki-
vantstructureand conformation, yetform opticallyclear neticdata showing that pancreatic lipase specific activity
micellar solutions, making them favorable for conventional dependsonfatty acyl chainlength of lecithin as well as
physical techniques (6-9, 24, 25). Their ability to solubilize triglyceride. Pancreatic lipase alone does not bind to lecithin
triglycerides makes them particularlyuseful for lipasestudies. surfaces (28). If the enzyme interacts with core triglycerides,
In contrast, long chain phosphatidylcholines mixed with tri- the lecithin chain length dependenceshould be small and the
glycerides tendtophase-separate, formingaphospholipid same for hydrolysis of tributyrin and trihexanoin. If triglyc-
bilayer with a small amountof triglyceride incorporated, and eride molecules are also present at the surface, thenthe
triglyceride oil. lecithin activity dependencecould reflect thermodynamic par-
Several reasonable models for the short chain lecithin/shorttitioning of the triglyceride between the core and surface in
chain triglyceride particles can be proposed. The simplest each mixed particle. This will depend on alkyl chain lengths
model, that thetriglyceride molecules are present among the and on micelle sizes,
lecithin molecules at themicelle interface, is inconsistent with Lipase Hydrolysis of Micellar a n d Monolayer Triglycer-
(i) large differences between alkyl chain TI values for 2 mol- ide/Lecithin Systems-Detergents in general are very strong
ecules forming a single mixed hydrophobic region, (ii) Mn2+ inhibitors of pancreatic lipase (28).Where they do not bind as
titration studies, and (iii) phospholipase activity. The Mn2+ micelles to thelipase, it hasbeen suggested that theinhibition
effect on triglyceride carbons is significantly smaller than its arises from the detergent interacting with the triglyceride
effect on lecithin alkyl chain carbons or buffer. This suggests emulsion interface. The structural view of the particles from
that most of the triglyceride does not reside at the particle NMR and enzyme studies places the triglyceride in a core
surface. Both phospholipase A2 (20) and phospholipaseC (15) with some small amount at the surfacein fast exchange.
show surface dilutionbehavior, i.e. hydrolysis dependson Although further kinetics and particle characterization will be
both bulk and surface phospholipid concentrations. The rate necessary to understand the observed dependence on both
of hydrolysis is directly proportional to the mole fraction of triglyceride and lecithin fatty acyl chain length in detail, we
lecithin (26). Extrapolation of the surface dilution shown by can compare these preliminarykineticswith other lipase/
phospholipasehydrolysis of dipalmitoyl-PC/Triton X-100 triglyceride assay systems.
mixed micelles (where the TritonX-100 has been shown tobe In a recent study of lipase activity toward didodecanoyl-
a neutral surface dilutor (27)) predicts a rate decreaseof about PC/trioctanoin monolayers, an optimalenzyme specific activ-
50%for the 1:l dioctanoyl-PC/tributyrin mixed micelles. Both ity about twice that of the triglyceride alone was obtained
enzymes show unaltered activityin the presence of tributyrin. around (0.7:l.O) lecithin/triglyceride(29). Surface (26) or
Comparison of diheptanoyl-PC/hexanol and diheptanoyl-PC/ “substrate” (29) dilutionwould predict that thespecific activ-
trihexanoin (4:l) mixtures further suggests the triglyceride is ity decreases with added lecithin. The monolayer kinetics for
not all a t t h surface.
e When a diheptanoyl-PC/hexanol sample lecithin/triglyceride 5 1.0 thenargue for analteration in
is prepared witha concentration of hexanol chains equivalent enzyme minimal specific activity (possibly an increase in kc,,
totrihexanoinfatty acyl chains (Le. 3 timesthenormal or decrease in K,,,).For lecithin/triglyceride > 1.0, a decrease
trihexanoin concentration), a stable, milky white emulsion is in enzyme specific activity was observed. Monolayer data is
formed.Hexanol, presentattheinterface because of the more difficult to obtain for these ratios since, as the number
hydrophilic character of the alcohol moiety, alters micelle of triglycerides decreases, enzyme bound to the surface de-
packing so that a larger particle is formed. creases (i.e. the concentrationof triglyceride is varying as well
In a second model for the particle, the triglyceride forms a as the ratiolecithin/triglyceride). The activity decrease could
hydrophobic core,which rigorously excludes short chain leci- be due toa further alteration in minimal specific activity or to
thin mnlecules. The existence of a triglyceride core is sup- a substrate dilution effect.
2722 Lecithin/
Chain
Triglyceride
ShortMicelles
Mixed
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 unalteredwith additional
lecithin. The first suggests that core triglyceride is the site of
lipase hydrolysis or that, if surface triglyceride is the prefer-
ential substrate, surfacebinding 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 tolipase. Such mixing behavior is atypical of most mixed
micelles containinglipid (30) butcannot beeliminated 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 ourmixed lipid particles.
Model “MiniZipoproteins”-Mixed lipid particles using
short chain lecithins as the micellar matrix can be exploited
further asa 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 formmicelle
particles with several compositional degrees of freedom. Ef-
fects of added componentson lecithin and triglyceride behav-
ior can be characterized by ”’C NMR and lipase/phospholi-
pasesusceptibility; theentireparticles can be probed by
quasielastic light-scattering and Raman scattering. essence,
In
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-
search located at the Francis Bitter National Magnet Laboratory,
Massachusetts Institute of Technology,
’ R. A. Burnsand M. F. Roberts, unpublished
results.
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