GASTROENTEROLOGY 78:954-962, 1966

Importance of Phospholipids, Pancreatic

Phospholipase AZ,and Fatty Acid for the
Digestion of Dietary Fat
In Vitro Experiments with the Porcine
Enzymes

BENGT BORGSTROM
Department of Physiological Chemistry, University of Lund, Lund, Sweden

Long chain triglycerides emulsified with phospho- The adult human consumes = 2-4 g phospholipid/
lipid are not directly available for hydrolysis by pan- day mostly phosphatidylcholine (PC)’ which func-
creatic lipase in vitro even in the presence of bile tions as an emulsifier of the more abundant tri-
salts and colipase. The inhibition can be overcome glyceride fat. In the small intestinal lumen, about 11
by pancreatic phospholipase A,. There is a limited g phospholipid, mainly PC, is mixed in the form of
hydrolysis of the phospholipid during this period. mixed bile salt micelles with the contents.’
The inhibition is explained by the finding that lipase It has been reported3 that PC inhibits the in vitro
does not bind to triglyceride emulsified by phos- hydrolysis of triglycerides as catalyzed by pancre-
pholipid but remains in the aqueous phase. A limited atic lipase. The activity of pancreatic phospholipase
hydrolysis of the phospholipid by phospholipase A, A, (PLA,) therefore may be important for the con-
results in the binding of lipase to the substrate inter- certed action of the pancreatic lipolytic enzymes on
face and a rapid rate of hydrolysis of the tri- dietary fat.
glyceride. With time the inhibition of lipase activity The present paper presents in vitro studies of the
can also be overcome by pancreatic lipase. A lag effect of phospholipid on hydrolysis of long chain
phase is seen before the accelerated hydrolysis of triglyceride by porcine pancreatic lipase. As pre-
triglyceride reaches a high rate. The length of the lag viously demonstrated, phospholipid inhibits hydrol-
phase is dependent on factors such as lipase and ysis of the triglyceride.” This inhibition can be over-
colipase concentration, pH, Ca++, and concentration come by PLA,. It is furthermore found that lipase
of bile salt. During the lag phase no significant hy- under certain conditions can also overcome this in-
drolysis of phospholipid occurs. The primary factor hibition and results are presented which throw light
is the binding of colipase to the substrate interface. on the mechanism of hydrolysis of triglyceride in
Fatty acid present in the oil phase or produced from the presence of phospholipid.
it by a limited hydrolysis of phospholipid by phos-
pholipase A, or triglyceride by lipase, changes the Materials and Methods
properties of the interface so that colipase can bind
and thereby lipase via its binding to colipase. The Chemicals
milieu of small intestinal content favors the con- Conjugated bile salts, taurodeoxycholate (TDC),
certed action of several factors to make dietary tri- glychodeoxycholate (GDC), taurocholate (TC), and gly-
glyceride available for an effective hydrolysis by cocholate (GC) were synthesized in this laboratory and
pancreatic lipase. were >98% pure as indicated by thin layer chromatogra-
phy (TLC). Egg PC was purified in this laboratory from
fresh eggs and showed one spot on TLC.’ Commercially
obtained chemicals were reagent grade. Intralipid. was a
product of Vitrum (Stockholm, Sweden). It is available as
Received June 19,1979. Accepted November 27.1979.
Address requests for reprints to: Dr. B. BorgsWm, Department
an emulsion which contains 100 or 200 g fractionated soy-
of Physiological Chemistry, POB 750, S-220 07 Lund, Sweden. bean triacylglycerol (TG), 12 g egg PC, and 25 g glycerol/
This study was supported by grants from the Swedish Medical loo0 ml. One milliliter of 29% Intralipid contains -225
Research Council. pmol TG and 15 pmol PC. The particle diameter is reported
0 1980 by the American Gastroenterological Association to be -2399 A.” Based on these values it can be calculated
0016-5065/60/050954-09502.25
May 1980 PHOSPHOLIPID. PHOSPHOLIPASE A,, AND IMPORTANCE FOR LIPASE 955

that 1 ml of 20% Intralipid contains -5 X lOI3 emulsion phase of the Intralipid emulsion was determined after
particles with a total interfacial are of =6 X 160 A.” Based high-speed centrifugation (-18,000 g) for 5 min in an Ole
on the reported surface area of PC’ of 75 A,’ 8 X lo*’ mole- Dick microcentrifuge (Nino Laboratory, Stockholm, Swe-
cules of PC would be an adequate concentration to cover den). A creamy top-phase and a slightly turbid aqueous
the surface as a monomolecular film. This corresponds to phase was obtained, the latter being sampled after the top
12. pm01 of PC of a total of 15 pm01 present. phase had been removed by suction. Lipase and colipase
14C-TG labeled Intralipid was a gift from Vitrum. Thin activities were determined as previously described using
layer silicic acid radiochromatography (TLC) using the tributyrin as the substrate.’
solvent system hexane/ethyl ether/methanol/acetic acid
45:10:1.5:1 was 96% TG, 0.6% diglyceride, 0.8% fatty acid.
The remaining lipid was more polar and remained at the
Results
origin. After centrifugation of labeled Intralipid, it was de-
termined that 35% of PC and 6% of radioactivity were Intralipid as a Substrate for Pancreatic Lipase
present in the aqueous phase. The radioactivity in the
When Intralipid diluted in buffer (150 mM
aqueous phase was more polar than triglyceride. We
NaCl, 1 mM CaCl,, 2 mM Tris-HCl, pH 8) or buffer
therefore concluded that almost all the TG of the Intra-
containing 4 mM TDC was added by lipase (final
lipid and approximately 65% of the PC are present in the
oil phase. The PC in the aqueous phase is present as lipo- concentration 10~' M), a very slow rate of titration
somes which do not contain significant amounts of other occurred. When colipase was included with 4 mM
lipids. The PC present in the aqueous phase may be re- TDC, a reaction rate as illustrated in Figure 1 was
sponsible for its slight turbidity. Emulsions in buffer were observed. From an initial rate of titration which was
also made in the laboratory by sonication after dissolving 0.03 pmol/min the rate slowly accelerated to 3.2
PC in triolein (TO) using a Branson sonifier (Branson pmol/min after 60 min. This lag phase was followed
Sonic Power Co., Danbury, Conn.). The particle distribu- by a sharp increase to 15.4 pmol/min. The rapid rate
tion in these emulsions were less homogeneous than that
of titration represents mainly hydrolysis of tri-
found in Intralipid.
glyceride to diglyceride, as indicated by radiochro-
matography of the products. This was followed in
Protein time by a slow rate of titration (< 1 pmol/min) repre-
senting the further hydrolysis of mainly di- to mono-
Porcine pancreatic lipase B (mol wt 52,000) and
colipase II (mol wt 11,000) were purified as previously de- glyceride. The results indicate that under the condi-
scribed.7 Porcine pancreatic PI& was a gift of G. H. de tions of the experiments Intralipid is not directly
Haas (Utrecht, The Netherlands). Porcine bile was ob- available as a substrate for lipase during the lag
tained from the local slaughter house. Lipolysis was deter- phase. The length of the lag phase, however, varies
mined titrimetrically using a Mettler automatic titration with the experimental conditions as will be dis-
system (Mettler Instruments A.G., Zurich, Switzerland). cussed later. No 1ysoPC was formed by lipase under
One-tenth to two milliliters Intralipid was diluted to 10 ml the conditions of the experiments.
with buffer 150 mM in NaCl, 2 mM in Tris-HCl, and other
additions as given in each experiment. Hydrolysis was
measured at ~IY’C by titration with a l-ml burette and 0.2 Intralipid as a Substrate for PLA,
M NaOH. The percent long chain fatty acid titrated at pH In the absence of bile salt the hydrolysis of
8,7, and 6 were 72,50, and 18, respectively. In a few exper- PC by PLA, was slow and incomplete. In the pres-
iments the products of lipolysis were determined using
ence of bile salt PLA, hydrolyzed the PC of Intra-
“‘C-labeled Intralipid, the separation was performed on 12
lipid, the rate and extent of hydrolysis was related to
x 12-cm silicic acid TLC plates using the solvent system
previously mentioned. Radioactivity was determined by the concentration of PLA, (Figure 2).
liquid scintillation in a Packard spectrometer (Packard In- The hydrolysis of PC by PLAZ, in the presence of
strument Co., Inc., Downers Grove, Ill.) after the spots had bile salt, had a dramatic effect on the lipase cata-
been indicated by iodine vapor and scraped off. Hydroly- lyzed hydrolysis of the triglyceride of Intralipid in
sis of PC to 1ysoPC in the Intraiipid or other emulsions the presence of colipase. The lag phase decreased in-
was determined after partitioning the phospholipids in a crease of PLA, concentration and the break in the
two-phase system formed by adding 206 ~1 of the in- lipase curve occurred when only a limited hydroly-
cubation medium to 4.0 ml solvent mixture (140 ml meth- sis of the PC had taken place (Figure 2). The titration
anol, 135 ml heptane, and 125 ml chloroform), mixing, and
curves include fatty acid hydrolyzed from both PC
then adding 1.05 ml H,O. In this system PC remains in the
and TG. With increase in PLA, and/or time with the
lower phase while 1ysoPC distributes 8:2 between the
phases. After separation of the phases, 50-200 ~1 aliquots
PC of Intralipid can be completely hydrolyzed. Fig-
were used for determining total-P according to the proce- ure 3 indicates a linear relationship between l/lag
dure of Itya and Ui’ as modified for lipid-P.’ The values time and concentration of PLA,. The rate of TG hy-
for 1ysoPC were corrected for the 8:2 partition. drolysis after the lag phase was independent of the
Distribution of lipase between the aqueous and oil concentration of PLA,.
956 BORGSTROM GASTROENTEROLOGY Vol. 78, No. 5, Part 1

Figure 1. General course of the lipase catalyzed hydrolysis of Intralipid. One milliliter of Intralipid (- 225 ~01 TG) was diluted to 10 ml
with buffer (150 mM NaCl, 1 mM Ca++, and 2 mM Tris-HCl pH 8). Lipase and colipase were added at zero time to final con-
centration of 0.5 X lo-’ and lo-’ M, respectively. The sample titrates slowly at an accelerating rate. After 75 min the rate of
titration changes abruptly and goes over to a high rate. The amplification of the recorder is changed as indicated. The reaction
then shifts to a slow phase when -140 gmol NaOH has been added corresponding to =190 pm01 fatty acids released out of a
total of 450 in primary ester bonds.

With PC-stabilized emulsions of TO sonicated in Factors Other than PLA, that influence the
buffer, which we had prepared, similar results were Lag Phase
obtained as with Intralipid. These emulsions when
sonicated in bile salt solution demonstrated a high As mentioned earlier the lag time of hydroly-
rate of hydrolysis in the presence of lipase + coli- sis of TG by lipase can be influenced by factors
pase without a lag phase. The explanation for this other than by PLAz, and it therefore became of inter-
difference is unknown. est to study which factors were responsible for this

a 0 0 0
III II H
Figure 2. Effect of PLA, on the hydroly-
_% sis of (A) Intralipid PC (for-
3 mation of 1ysoPC = LPC) and
- 8 on the lipase catalyzed hy-
- ??
‘~~_____““““” __~,_________________._________________ drolysis of the Intralipid TG
E (0). One milliliter Xi% Intra-
- $ p’ lipid was diluted to 10 ml; fi-
nal concentration of buffer:
150 mM NaCl, 1 mM Ca++, 2
mM Tris-HCl pH 8.0. and 4
mM TDC. Incubated at 4O“C
and titrated. Lipase and coli-
pase were added at zero time
to a final concentration of
lo-’ M and 2 X lo-’ M. The
final concentration of PLAz
were: I, 10 &ml; II, 5 p&ml;
III, 2.5 pg/ml, and IV, 1 pg/ml.
PC = 1.24 pmol/ml.

1 6 10 16 MINUTES
May 1980 PHOSPHOLIPID. PHOSPHOLIPASE A,, AND IMPORTANCE FOR LIPASE 957

Co++ lons

In the absence of Ca++ the lag phase was in-

0 definitely long; increase in Ca++ decreased the lag
time (Figure 4B).
ii
01 F

v
I: Lipase and Colipase
<
r As previously discussed colipase is necessary
0
for lipase to hydrolyze the Intralipid triglyceride. In-
05 0.05 crease in colipase decreases the lag phase. In Figure
4C the effect of a parallel increase in lipase + coli-
pase is shown to decrease the lag phase and also to
increase the rate of triglyceride hydrolysis after the
I1 III,,.,,.
lag. Preincubation of the system with colipase fol-
0.5 1.0 lowed by lipase had no effect on the lag time and
5.0 10.0 : preincubation of the emulsion with lipase had only a
LIB PLA. ml-’
marginal effect on the lag time after the addition of
Figure 3 l/Lag time in relation to PLA, concentration. One-half colipase (due to the inherent colipase activity of lip-
milliliter of 20% Intralipid was diluted to 10 ml with ase). The pH of the bulk phase had a strong influ-
standard buffer 4 mM in TDC pH 8.0, 40°C. At zero
time lipase and colipase final concentrations 2 x lo-’
ence on the lag time. In Figure 6 the reciprocal of the
and 4 x lo-’ M were added. Five min later PLAz was lag phase shows an optimum around pH 6.5. The fig-
added to give the final concentrations given on the x- ure includes the pH curve for two different concen-
axis. The lag time was measured from the addition of trations of Ca++.
PLA, until the break occurred. In the different situations discussed above a slow
and accelerating rate of hydrolysis of ester bonds oc-
lag time in order to understand the mechanism of re- curred during the lag phase, and the length of the lag
versal of the lag phase. phase was dependent on this rate. It appears that a
certain critical extent of hydrolysis has to be
reached before the lag phase changes over to a high
Bile Salts
rate of hydrolysis of triglyceride. The limited and
The lag phase was decreased by an increase slow rate of hydrolysis of the TG during the lag
in concentration of TDC (Figure 4A). This effect was phase as indicated by titration figures has been veri-
parallel to an increased partitioning of PC to the fied by directly measuring the product formation
aqueous (micellar) phase (Figure 5). The trihydroxy- from [‘YZ]triolein. With 410 pmol of TG (0.5 ml In-
conjugates taurocholate and glycocholate corn ple- tralipid) the critical hydrolysis for the break to occur
tely prevented the activation of Intralipid by lipase is 5-7 pmol fatty acid liberated, corresponding to a
+ colipase in the concentration range 2-16 mM at concentration in the incubation medium of 5-i’ x
pH 6.5 and 6.0. PLA2, however, activated the lipase low4M fatty acid. The break in the lag phase is de-
system also under these conditions. pendent on the activity of pancreatic lipase against

Figure 4. Effect of bile salt (TDC) (A), Ca++ (B),

and (lipase + colipase) (C) on the lag
time and the rate of hydrolysis of Intra-
lipid-TG by pancreatic lipase. One-half
of a 20% Intralipid was diluted to 10 ml
in buffer (150 mM NaCI, 2 mM Tris-HCI)
with the different additions as shown. In
A, Ca++ was 6 mM; TDC, 4 mM. In B,
TDC was 4 mM; and in C, Ca++ was 6
mM; TDC, 4 mM. The amounts of lipase
and colipase added in A and B gave a fi-
nal concentration of lo-’ M and 2 x lo-’
M. In C, a mixture of lipase (2 x 10W5M)
and colipase (10M4M) in the relation 5 : 2
were added.
958 BORGSTRC)M GASTROENTEROLOGY Vol. 78, No. 5, Part 1

100 -

t
PC

5 .
RADIOACTIVITY

r
a
0 2 4 TDC “M] 12
i [’ ’
Figure 5. Partitioning of PC and radioactivity between oil and
aqueous phases of Intralipid diluted into buffer with
7
varying (TDC). One-tenth milliliter, 10%
[W]trioleoylglycerol labeled Intralipid was diluted in
0.80-ml buffer with varying (TDC). After 1 hr at room
temperature the samples were centrifuged for 30 min
in a Dick centrifuge. The fat pellet was removed and
the aqueous phase was sampled for radioactivity and
for the determination of PC as described in Methods. 0 0.1 02
The figures are given as percent PC (0) and radio-
ml PORCINE BILE ml’
activity (A) present in the aqueous phase.
Figure 7. Effect of porcine bile on the lag time and the rate of
the TG of the substrate and in no cases have any hy- TG-lipolysis following the lag phase at different con-
drolysis of PC been seen to occur with this enzyme. centrations of PLA,. Five tenths milliliter of 20% Intra-
lipid was diluted to 10 ml with the standard buffer 4
The course of the reaction is, however, very similar
mM TDC and porcine bile added as given. The titra-
to that caused by PLA, and previously discussed. tion was run at pH 8.0 and 40°C. Lipase and colipase
final concentration 2 x lo-’ and 4 X 10-s M were
Inhibition of Lipolysis with Porcine Bile and added at the start. After 5 min incubation PLA, was
Release of Inhibition by PLA, added at three different levels: 00, 1.0; ??0, 2.0; and
VA, 4.0 pg/ml final concentration.
Porcine gallbladder bile was found to be an
inhibitor to Intralipid hydrolysis by lipase + coli- pase. Figure 7 shows the effect of 0.1 and 0.2 ml bile
(phospholipid (PL) concentration of the undiluted
bile = 21.0 pmol/ml) added to a final volume of lo-ml
incubation system on the lag time and the rate of TG
hydrolysis after the lag time at three different con-
centrations of PLA,. The lag time is increased when
the bile concentration is increased and is decreased
with increase in PLA, concentration. The high rate
of TG hydrolysis is not affected by bile. In the exper-
iments in which 0.1 ml bile/l0 ml was used with
varying amounts of PLA, (Figure 8) the samples for
analysis of 1ysoPL were taken in the same sequence
of the titration curve, although the lag phase had a
different length in the different experiments. The re-
sults indicate that the break, i.e., the end of the lag
phase occurred at approximately the same concen-
tration of 1ysoPL. At that point some 20% of the PL
5 6 7 0 9 PH had been hydrolyzed. PLA, efficiently hydrolyzed
Figure 6. Effect of pH of the bulk phase on the lag time for TG PL in diluted porcine gallbladder bile (Figure 9).
hydrolysis of Intralipid by pancreatic lipase. One-half
milliliter of 20% Intralipid was diluted to 10 ml with
buffer containing 4 mM TDC at 1 mM (A) and 8 mM Distribution of Lipase and Colipase During
(0) (Ca”). The pH was adjusted to the figures in the Reaction with Intralipid
question in the pH stat. Final lipase and colipase con-
centrations 0.5 x lo-' and low7 M, respectively. Fig- The distribution of lipase between the aque-
ures were given as l/lag time. ous phase and substrate interface during the lag
May 1980 PHOSPHOLIPID. PHOSPHOLIPASE A,, AND IMPORTANCE FOR LIPASE 959

040
100

I
/ i
030
I
??
::
0
??BILE f

d* 0
0.20
50 0

I 010
0 ??
0 a
/.
x 0
i: t,
z 0 2 10 20

f' 0.30 1 ,ug PLA, ml’

I NO BILE
Figure 9. Percent hydrolysis of porcine bile PL by porcine PLA,.
Gallbladder bile was diluted 1: 5 with Tris-HCI pH 6.5,
6 mM in Ca++. Eight-tenths milliliter of the dilution
was added to small glass tubes, added with PLA, to a
total volume of 1.0 ml, and incubated for 20 min at
40°C. LysoPL was determined as described in Meth-
ods. The initial concentration of total bile P was 4.5 x
10e3 M; of this, 12% distributed initially to the upper
b phase (Pi + 1ysoPL) and was corrected for.
0 1 2 3 4 5 TIME

T T T T T T was found in the aqueous phase. Samples were then

taken at intervals as shown in Figure 10B. With time
there was an accelerated increase in the partition of
lipase to the emulsion interface that was parallel to
Figure 8. Incubation consisted of 0.5 ml 20% Intralipid in 10 ml
total volume of 4 mM TDC (150 mM NaCI, 1 mM
an increase in the rate of hydrolysis until the second
Ca++, and 2 mM Tris-HCl pH 8.0). PC = 0.71 pmol/ml. phase of hydrolysis was reached. The distribution of
In the upper graph O.l-ml porcine bile had been added
and the total PL was 0.92 pmol/ml. Thus 0.21 pmol
was from bile. Three different levels of PLAz were
added: 10 (0) 20 0, and 40 (A) pg. respectively. Sam
ples for analysis were taken at different points in the
titration sequence indicated.

phase and after the addition of PLA, was investi-

gated. Samples were taken at different times after
addition of lipase + colipase and PLA, at pH 8.0 and
the oil phase separated from the aqueous phase by
high-speed centrifugation as described in Methods.
The lipase content was determined enzymatically in
the aqueous phase. The results are given in Figure
10A. They indicate that during the lag phase more A B
than 95% of the lipase is in the aqueous phase in the
0 5 10 15 30 0 5 10 16 30 45 MIN
presence of colipase. When the break in the titration
Figure 10. Distribution of lipase (0) and cohpase (A) to the
curve is obtained after addition of PLA2, lipase
aqueous phase when fatty acids were generated by:
leaves the aqueous phase suggesting that it is now A. PLA, in the presence of lipase and colipase, and
bound to the substrate interface. The low activity of B. Lipase and colipase. Conditions of the experi-
lipase against Intralipid during the lag phase is thus ments: A. 4 mM TDC, 1 mM Ca++, pH 8.0 final con-
centration of lipase = 2 X lo-" M, cohpase = 3 X lo-'
explained by the finding that lipase in the presence
M, and PLA, = 1 X lop7 M. Lipase + colipase was
of colipase and in bile salt solution does not bind to added from the start. PLAz was added 14 min later.
the Intralipid interface. After the rapid phase of hy- B. 4 mM TDC, 1 mM Ca++, pH 7.5. Final concentra-
drolysis more lipase is again found in the aqueous tion of lipase = 2 x lo-* M; colipase 3 x lo-“ M. Coli-
phase. pase was added at zero time followed 5 min later by
lipase. Sampling was done at 5 min intervals. The
A similar experiment was also performed, in
lage time was 26 min. Samples of 0.7 ml were taken
which colipase was added to the incubation mixture as indicated and immediately centrifuged to give an
at pH 7.4 followed by lipase (Figure 10B). In the first oil and an aqueous phase. The latter was sampled
sample taken 2 min later -100% of the lipase activity and used for determination of lipase and colipase.
960 BORGSTROM GASTROENTEROLOGY Vol. 78, No. 5, Part 1

lipase to the oil phase thereafter decreased some- Discussion

what. The course of the reaction is very similar to As was previously demonstrated by Klein et
that seen when the lag phase was reversed by PLA, al.” PC inhibits TG hydrolysis by pancreatic lipase.
in the previous experiment. In these experiments we These authors also found that the inhibition could
measured also the colipase partition, and the results be reversed by increasing the lipase concentration.
are included in Figure 10B. It is apparent that coli- They suggested that PC formed an external phase of
pase initially stays in the aqueous phase and does the emulsified TG droplet and thereby prevented ac-
not bind to the Intralipid interface. cess to the lipase; they did not speculate in what
The generation of fatty acids from PC in the ab- way an increase in lipase concentration could over-
sence of lipase results in a binding of colipase to In- come the inhibition.
tralipid in the same manner as in the experiments in In the present study most of the experiments
Figure 10B. In these experiments shown in Figure 11, were performed with a commercially available
colipase, PLA,, and lipase were added in sequence to fat emulsion Intralipid that is stabilized by purified
Intralipid in dilution with bile salt containing buffer egg phospholipid which is mostly PC. The molar
and their effects on colipase and lipase distribution ratio TG/PC is 15:l and it was calculated that with
to the aqueous phase determined. Colipase alone the particle size of the emulsion there will be more
stayed in the aqueous phase; addition of PLA, re- than enough PC to cover the interface of the TG
sulted in the uptake of 2: 50% of the colipase into the droplets by a monolayer and also that some PC may
emulsion interface. Subsequent addition of lipase re- be present as liposomes. The Intralipid is homoge-
sulted in a further uptake of colipase to the oil phase neous in particle size and allows calculation of dif-
that was parallel to an uptake of lipase to the same ferent parameters of interest. Essentially similar re-
phase and a rapid hydrolysis of TG. sults were obtained by laboratory made emulsions
which are less homogeneous in particle size.
The inhibition of lipolysis by PC could be ex-
Effect of Oleic Acid on the Lag Phase and on
plained by the finding that lipase stayed in the aque-
the Binding of Lipase to the lntralipid
ous phase even in the presence of bile salt and coli-
Substrate
pase. The inhibition by PC could be overcome by
Varying quantities of oleic acid were added to pancreatic PLA,, and the length of the lag phase in
the titration vessels, and 10 ml of 4 mM TDC in buf- the presence of lipase was related to the concentra-
fer were added at different pH values. The solutions tion of PLA,. Analysis of the amounts 1ysoPL formed
were stirred for several minutes to obtain a solution indicates that the TG interface becomes available to
of mixed oleic acid bile salt micelles in the concen- lipase after only a limited hydrolysis of PC.
tration range of 0.02-lpmol fatty acid/ml. The Intra- The lag phase of Intralipid could also be overcome
lipid was added followed by colipase and lipase. The by lipase (in the presence of colipase). This effect
results are given in Figure 12 and show that the pres- was, however, not a consequence of PC hydrolysis
ence of oleic acid decreases the length of the lag by lipase but due to a limited accessibility of the In-
phase at any given pH and that the lag phase is tralipid-TG to lipase. The fatty acids initially formed
shorter the lower the pH at the same oleic acid con- favor the binding of lipase to the interface; the rate
centration. of hydrolysis is accelerated until. at a critical con-

Figure 11. Effect of fatty acid generated by PLA,

on the distribution of lipase (0) and
colipase (A) to the aqueous phase of In-
tralipid. Intralipid 0.75 ml was diluted
to 15 ml with a bile salt containing buf-
fer (4 mM TDC, 150 mM NaCl, 1 mM
Ca++, 2 mM Tris-HCl, pH 7.5). Colipase
to give a final concentration of 3 X 10e8
M was added at C. After 15 min PLAz
6
was added to a final concentration of 5
X lo-’ M. At L lipase was added to a fi-
0 nal concentration of = 1.5 x 10e8 M.
t Samples were taken at the times given
and analyzed for colipase and/or lip-
ase. The solid line shows the titration
to UIN curve. The sensitivity of the recorder
C L
Lp was changed as indicated by the broken
A2 line.
May 1950 PHOSPHOLIPID, PHOSPHOLIPASE AZ, AND IMPORTANCE FOR LIPASE 961

salt was shown to dissolve PC into the aqueous

phase probably in mixed micelles, thereby decreas-
PH
ing the PC concentration at the interface. Increase in
A 80 lipase and colipase concentration as well as Ca++
0 7.5 can be expected to increase TG hydrolysis and
v 7.0 therefore fatty acid liberation. In line with our re-
?? 6.5 sults is the finding that the binding of lipase and
colipase to siliconized glass beads is hindered by
pretreatment with biliary lipids.‘”
Phosphatidylcholine used in this investigation
was not hydrolyzed as liposomes in the temperature
range 25°-40”C in agreement with previous experi-
ments;” addition of bile salt to the liposomes af-
fected hydrolysis. The PC of Intralipid was hydro-
lyzed by PLA, in the absence of bile salt but only
slowly. Bile PL could be efficiently hydrolyzed by
PLA, in contrast to an earlier report that they were
resistant to the enzyme.” In no case, however, was
PC of Intralipid hydrolyzed to any measurable ex-
0.1 05 1
tent by pancreatic lipase in contrast to an earlier re-
FATTY ACID x lO-’ M
port on the effect of this enzyme on the fatty acid in
Figure 12. Effect of added fatty acid on the lag phase for Intra-
lipid TG hydrolysis by lipase in the presence of coli-
the l-position of PC.‘” A finding that is not under-
pase. Oleic acid were added to the titration vessels to stood at the present time is that Intralipid in dilution
the final concentrations given, 9.5 ml of buffer (4 mM with TC or GC in the concentration range 2-15 mM
TDC, 1 mM Ca++, 150 mM NaCl, and 2 ml Tris-HCI) was not accessible to lipase. PLAZ, however, hydro-
were added, and the fatty acid was brought in mixed
lyzed PC even with the trihydroxy- bile salts and re-
micellar solution by stirring. pH was adjusted to the
values given in the figure, and 0.5 ml Intralipid was
versed lipase inhibition.
added followed by colipase and lipase to give the fi- The present experiments have in general been de-
nal concentrations lo-’ and 0.5 X lo-' M. The lag signed to approximate the conditions of intestinal
time was measured and plotted over oleate concen- contents in vivo as to fat (lo-20 mg/ml) and enzyme
tration.
concentration (l-2 X low7 M of lipase and colipase).‘4
In most of the experiments pH 8.0 was used due to
the fact that the fatty acid are more completely tit-
centration of fatty acid, lipase is optimally bound rated and also because the lag phase is more marked
and hydrolysis takes place at a rate related to the at this value. When considered important the whole
lipase concentration. pH range 6.5-8 has been studied. The calcium con-
A crucial question for the interpretation of the centration has in most experiments been kept at 1
mechanism of the PC inhibition and its reversal by mM, but also higher values have been used closer to
PLA, or lipase is if PC hinders colipase binding to those to be expected in vivo (7 mM in human duode-
the emulsion interface. The results obtained (Figure nal content after breakfast).15
11) definitely show that colipase cannot bind to In- The molar ratio PC/TG in most of the experi-
tralipid and that the primary effect of the fatty acids ments was 1:15 compared with the average in the
generated by PLA, is to make possible the binding of human diet which can be calculated to be =1:40.l
colipase to the substrate interface. Lipase then binds Some particular food items, as egg yolk, however,
to colipase at the interface in a position favorable for has a much higher ratio. The more important admix-
catalysis of TG hydrolysis. The results in Figure 11 ture to the dietary fat of PL takes place via the bile
speak in favor of a cooperative binding of lipase and and the molar ratio calculated from analysis of in-
colipase to the fatty acid generating interface. The testinal contents in humans after a test meal is in the
elucidation of the mechanism by which fatty acid order of 1: 10.‘5.‘6
affects the binding of colipase to the substrate has to The present experiments have to be regarded as a
await further experiments; it seems, however, defi- study of single factors responsible for a concerted
nitely to be dependent on the pH and therefore on reaction to hydrolyze dietary fat in the small in-
the charge of the system. Several different factors testinal contents. The results indicate that under
were operative to influence the lag phase. A com- physiologic conditions little inhibition of lipase by
mon mechanism seems to be to increase the accessi- dietary or bile PL would be expected. Several factors
bility of colipase for the substrate. Increase in bile will be operative to make the dietary TG rapidly
962 BORGSTROM GASTROENTEROLOGY Vol. 78, No. 5, Part 1

available for hydrolysis by lipase. Important will be pholipid-stabilized soybean oil emulsions. Int J Pharmaceut
the PLA2 derived by tryptic activation of the zymo- 1:141, 1978
8. Papahadjopoulos D: Phospholipids as model membranes:
gen in pancreatic juice. Already by a limited hydrol- monolayers, bilayers and vesicles. In: Form and Function of
ysis of the PC of intestinal contents enough fatty Phospholipids. Edited by GB Ansell, RMC Dawson, JN Haw-
acids are produced to trigger lipolysis of the tri- thorne. Amsterdam, Elsevier, 1973, p 143-189
glyceride at a high rate. Even in the absence of PLA,, 7. Borgstrom B, Erlanson C: Interaction of serum albumin and
other proteins with porcine pancreatic lipase. Gastroenterol-
conditions would be expected to be favorable for a
ogy 75:382, 1978
rapid “activation” of dietary TG by lipase. Dietary 8. Itaya N, Ui M: A new micromethod for the calorimetric deter-
fat reaching the duodenum contains some 1040% mination of inorganic phosphate. Clin Chim Acta 14:361,1966
free fatty acids? as an affect of the lingual lipase.‘” 9. Belfrage P, Wiebe T, Lundquist A: Methods for the determi-
This amount most probably is enough for an imme- nation in the nanomole range of lipids in liver fine-needle as-
piration biopsies. Stand J Clin Lab Invest 2653, 1970
diate strong binding of colipase to the glyceride sub-
10. Lairon D. Nalbone G, Lafont H, et al: Possible roles of bile
strate also in the presence of PL. In fact gastric lipo- lipid and colipase in lipase adsorption. Biochemistry 17:5263,
lysis (by lingual lipase!) has been shown previously 1978
to facilitate further lipolysis of milk fat by pancre- 11. Ihse I, Arnesjo B: The phospholipase A, activity of human
atic lipase in the presence of bile salts and coli- small intestinal content. Acta Chem Stand 27:2749, 1973
12. Boucrot P, Clement JR Resistence to the effect of phospholi-
pase.‘B.19
pase AZ of the biliary phospholipids during incubation of bile.
It has previously been shown that bile salts in the Lipids 6:652, 1971
presence of lipase and colipase reverse the inhib- 13. DeHaas GH, Sarda L, Roger J: Positional specific hydrolysis of
itory effect of pancreatic lipolysis caused by amphi- phospholipids by pancreatic lipase. Biochim Biophys Acta
philic dietary proteins.’ The gastrointestinal tract 100:638, 1965
14. Patton JS, Albertsson PA, Erlanson C. et al: Binding of por-
thus provides for a rapid and efficient hydrolysis of
tine pancreatic lipase and colipase in the absence of sub-
dietary fat by a concerted action of three enzymes, strate studied by two-phase partition and affinity chromatog-
one coenzyme, and bile salts. raphy. J Biol Chem 253:4195, 1978
15. Mansbach CM, Cohen RS, Leff PB: Isolation and properties of
the mixed lipid micelles present in intestinal content during
References fat digestion in man. J Clin Invest 56:781, 1975
16. Arnesj6 B, Nilsson A, Barrowman J, et al: Intestinal digestion
1. Borgstriim B: Phospholipid absorption. In: Lipid Absorption: and absorption of cholesterol and lecithin in the human.
Biochemical and Clinical Aspects. Edited by K Rommel, H Stand J Gastroenterol 4853, 1969
Goebell. Lancaster, England, MTP, 1978, p 65-70 17. Borgstrom B, Lundh G. Dahlquist A, et al: Studies of intestinal
2. Northfield TC, Hofmann AF: Biliary lipid output during three digestion and absorption in the human. J Clin Invest 361521,
meals and an overnight fast. Gut 161.1975 1957
3. Klein E, Lyman RB, Peterson L, et al: The effect of lecithin on 18. Hamosh M, Klaeverman HL, Wolf RO, et al: Pharyngeal lip-
the activity of pancreatic lipase. Life Sci 61305, 1967 ase and digestion of dietry triglycerides in man. J Clin Invest
4. Singelton WS. Gray MS, Brown ML, et al: Chromatographic- 55963. 1975
ally homogeneous lecithin from egg phospholipids. J Am Oil 19. Olivecrona T, Billstrom A, Fredrikzon B, et al: Gastric lipo-
Chem Sot 42:53,1965 lysis of human milk lipids in infants with pyloric stenosis.
5. Dawes VH, Groves MJ: The effect of electrolytes on phos- Acta Paediat Stand 62:520, 1973