Biochemical and Molecular Action of Nutrients

Phosphatidylcholine Inhibits and Lysophosphatidylcholine Enhances the

Lymphatic Absorption of ␣-Tocopherol in Adult Rats1
Sung I. Koo2 and Sang K. Noh
Department of Human Nutrition, Kansas State University, Manhattan, Kansas 66506

Downloaded from https://academic.oup.com/jn/article-abstract/131/3/717/4687210 by guest on 05 June 2020

ABSTRACT This study was conducted to compare the effects of enterally infused phosphatidylcholine (PC) and
lysophosphatidylcholine (lysoPC) on the lymphatic absorption of ␣-tocopherol (␣TP) in male rats. In expt. 1,
bile-diverted rats with mesenteric lymph cannulas were infused at 3.0 mL/h for 8 h with a lipid emulsion containing
5.0 ␮mol ␣TP, 565 ␮mol 14C-triolein (14C-OA) and 396 ␮mol Na⫹-taurocholate with 80 ␮mol 1,2-dipalmitoyl PC
(DPPC) or 1,2-dilinoleoyl PC (DLPC) or without PC (NoPC) in 24 mL phosphate-buffered saline (pH 6.6). In expt. 2,
the effects of 1,2-dioleoyl PC (DOPC) and 1-oleoyl-2-hydroxy-PC (lysoPC) on ␣TP and 14C-cholesterol absorption
were compared in rats with lymph cannulas. When DPPC or DLPC was infused, the lymphatic absorption of ␣TP
was lowered drastically. The cumulative absorptions of ␣TP in rats infused with DPPC and DLPC were 45 and 52%,
respectively, of the control values (NoPC). No significant difference was noted between the PC groups. In contrast,
the absorption of 14C-OA was increased by 42 to 43% in rats infused with DPPC or DLPC compared with that in
NoPC rats. Phospholipid outputs also were significantly higher in DPPC (34.0 ⫾ 5.5 ␮mol/8 h) and DLPC (32.4
⫾ 2.4 ␮mol/8 h) rats than in NoPC rats (21.2 ⫾ 4.2 ␮mol/8 h). When lysoPC was infused, the absorptions of ␣TP
and 14C-cholesterol were increased markedly compared with those for DOPC, with no significant difference in PL
output between groups infused with DOPC and lysoPC. These observations provide clear evidence that PC
present in a lipid emulsion inhibits ␣TP absorption, whereas it enhances the absorption of fat. The data also
demonstrate that lysoPC simultaneously increases the absorption of ␣TP and cholesterol. The findings indicate
that luminal PC inhibits the absorption of ␣TP and that hydrolysis of PC is critical to improving the intestinal
absorption of the vitamin. J. Nutr. 131: 717–722, 2001.

KEY WORDS: ● absorption ● ␣-tocopherol ● cholesterol ● lysophosphatidylcholine ● phosphatidylcholine

● rats

Phosphatidylcholine (PC)3 of biliary or dietary origin plays
an important role in the intestinal absorption of dietary fat and
fat-soluble nutrients. O’Doherty et al. (1) first demonstrated
that the deprivation of biliary PC results in the impaired
uptake and absorption of fatty acid (fat) and that the provision
of PC in bile-diverted rats not only restores the absorption of
fat but also stimulates protein synthesis during active fat ab-
sorption. This observation has been confirmed in subsequent
studies by other investigators (2,3). Growing evidence, how-
ever, indicates that PC may inhibit the intestinal uptake and
absorption of certain lipids. For instance, the presence of PC in
bile-salt micelles has been shown to inhibit cholesterol uptake
by intestinal segments and everted sacs under in vitro condi-
tions (4 – 8). A marked decrease in cholesterol absorption also
1
Supported by U.S. Department of Agriculature National Research Initiative
Competitive Grants Program (96-35200-3207) and the Kansas Agricultural Ex-
periment Station (KAES) (contribution 01-79-J).
2
To whom correspondence should be addressed. E-mail: koo@humec.ksu.edu
3
Abbreviations used: ␣TP, ␣-tocopherol; 14C-CH, 14C-cholesterol; 14C-OA,
14
C-oleic acid; DLPC, 1,2-dilinoleoyl phosphatidylcholine; DOPC, 1,2-dioleoyl
phosphatidylcholine; DPPC, 1,2-dipalmitoyl phosphatidylcholine; lysoPC,
1-oleoyl-2-hydroxy phosphatidylcholine; PC, phosphatidylcholine; PL, phospho-
lipid.
has been shown in humans infused intraduodenally with pu-
rified soy PC (9). More recently, a study with Caco-2 cells (10)
showed that PC in bile-salt micelles decreased the cell uptake,
esterification and secretion of cholesterol without affecting the
uptake of fat and monoacylglycerol, whereas the substitution
of lysophosphatidylcholine (lysoPC) for the PC or the addi-
tion of pancreatic phospholipase A2 (PLA2) reversed the
inhibition of cholesterol uptake by PC. In addition, PLA2
inhibitors or antibodies were shown to abolish the PLA2-
dependent increase in cholesterol uptake (10,11). The inhib-
itory effect of PC on cholesterol uptake also was observed
when PC was incorporated into lipid emulsions (12). The
initial hydrolysis of the surface PC in a lipid emulsion by
pancreatic PLA2 was required for hydrolysis of the core triac-
ylglycerol (TG) by pancreatic lipase/colipase and for the stim-
ulation of cholesterol uptake by rat intestinal cells.
Of particular interest is the observation that micellar PC
causes a drastic decrease in the intestinal absorption of ␣-to-
copherol (␣TP), a fat-soluble vitamin, as measured by using an
intestinal segment perfused in situ (13). At the present, direct
evidence for such an effect of PC is lacking from in vivo
studies using conscious animals. Furthermore, the exact mech-
anism underlying the inhibitory effect of PC on the intestinal

0022-3166/01 $3.00 © 2001 American Society for Nutritional Sciences.

Manuscript received 18 September 2000. Initial review completed 16 November 2000. Revision accepted 11 December 2000.

717
718 KOO AND NOH
uptake and absorption of ␣TP remains to be elucidated. Thus,
the present study was conducted to: 1) determine the effects of
PC differing in their fatty acid makeup on ␣TP absorption in
bile-diverted rats with lymph cannula and 2) examine whether
lysoPC increases the intestinal absorption of the vitamin.
MATERIALS AND METHODS
Animals and diet. Male Sprague-Dawley rats weighing 242 ⫾ 7 g
were purchased from Harlan Industries (Indianapolis, IN) and housed
singly in wire-bottomed plastic cages in a room of controlled tem-
perature (22–24°C) and lighting (light off: 0900 –2100 h). On arrival,
rats had free access to a nutritionally adequate AIN-93G diet (14)
containing egg white in place of casein (Table 1). All rats had free
access to deionized water throughout the study. Animals were cared
for in an animal care facility in the Department of Human Nutrition,
Kansas State University, that was fully accredited by the American
Association for the Accreditation of Laboratory Animal Care. Ani-
mals were maintained in accordance with the policies and guidelines
for animal care and use procedures of the Kansas State University
Institutional Animal Care and Use Committee.
Experiment 1
Cannulation of bile and lymph ducts. Rats weighing 348 –368 g
were divided into three groups of five rats each. After not being fed
for 15 h, rats were anesthetized with halothane using a vaporizer
(2.0% halothane in 2.0 L oxygen/min). Cannulation of the bile and
mesenteric lymph ducts was performed as described previously
(15,16). Briefly, after midline abdominal incision, the bile duct was
cannulated with PE-10 tubing (Clay Adams, Sparks, MD), which was
secured in place with suture (4-0 silk; Ethicon, Somerville, NJ).
Subsequently, the major mesenteric lymph duct was cannulated with
polyethylene tubing (SV 31 tubing; Dural Plastics, Auburn, Austra-
lia) and fixed with cyanoacrylate glue (Krazy Glue, Columbus, OH).
A silicone infusion catheter (Silastic medical grade tubing; Dow
Corning, Midland, MI) was inserted into the proximal duodenum via
the gastric fundus and secured with purse-string suture (4-0 silk;
Ethicon). The bile and lymph cannulas and the infusion catheter
were exteriorized through the right flank. The rats were placed in
individual restraining cages and allowed to recover for 20 h in a warm
recovery chamber maintained at 30°C. During the recovery period,
rats were infused via the infusion catheter with phosphate-buffered
saline (PBS) buffer (277 mmol glucose, 6.75 mmol Na2HPO4, 16.5
mmol NaH2PO4, 115 mmol NaCl and 5 mmol KCl per L, pH 6.6) at
3.0 mL/h via a syringe pump (model 935; Harvard Apparatus, South
Natick, MA).
TABLE 1
Diet composition1
Ingredient Amount
g/kg
Egg white 200.0
Corn starch 396.5
Dextrinized corn starch 132.0
Dextrose 100.0
Cellulose 50.0
Soybean oil2 70.0
Mineral mix 35.0
Vitamin mix 10.0
Biotin (1 mg/g biotin sucrose mix) 4.0
Choline bitartrate 2.5
1 Formulated and supplied from Dyets (Bethlehem, PA) according to
the recommendations of the American Institute of Nutrition (14).
2 Contained 0.02% tert-butylhydroquinone.
Composition and infusion of lipid emulsion. After postoperative
recovery, rats were infused at 3.0 mL/h with a lipid emulsion con-
taining 5.0 ␮mol ␣TP (all-rac-␣-tocopherol, 97%; Aldrich Chemi-
cal, Milwaukee, WI), 27.8 kBq [carboxyl-14C]triolein (14C-OA; spe-
cific activity, 3.8 GBq/mmol; DuPont-New England Nuclear, Boston,
MA), 565 ␮mol triolein (95%; Sigma Chemical, St. Louis, MO) and
396 ␮mol Na⫹-taurocholate with 80 ␮mol 1,2-dipalmitoyl PC
(DPPC; 99%; Avanti Polar Lipids, Alabaster, AL) or 1,2-dilinoleoyl
PC (DLPC; 99%; Avanti Polar Lipids) or without PC (NoPC) in 24
mL of PBS. During infusion of the lipid emulsion, lymph was col-
lected at hourly intervals for 8 h in preweighed ice-chilled plastic
tubes containing 30 ␮g n-propyl gallate and 4 mg Na2EDTA. Lymph
samples were stored at ⫺70°C for lipid analyses.
HPLC analysis of ␣TP in lymph. ␣TP was extracted with
acetone with a slight modification of the procedure (17). Briefly, 100
Downloaded from https://academic.oup.com/jn/article-abstract/131/3/717/4687210 by guest on 05 June 2020
␮L lymph was placed into a glass test tube, followed by the addition
of 1 mL acetone and 150 mg anhydrous sodium sulfate. The contents
were mixed vigorously on a vortex mixer. After centrifugation at
1000 ⫻ g at 4°C for 10 min, the upper phase was filtered through a
PTFE syringe filter (0.45 ␮m; Alltech Associates, Deerfield, IL), dried
under N2 and resolubilized in a defined volume of chloroform/meth-
anol mixture (1:3, v/v). ␣TP acetate was added as an internal stan-
dard into each sample. The extracts were separated with a Beckman
HPLC instrument with System Gold software (Beckman Instruments,
Fullerton, CA) equipped with a C-18 reverse-phase column (Alltima
C18, 5 ␮m, 4.6 ⫻ 150 mm; Alltech Associates). Methanol was used
as the mobile phase at 2 mL/min. Typical elution times were 4.1 min
for ␣TP and 5.3 min for ␣TP acetate. Detection was monitored at
292 nm (Module 166; Beckman Instruments). The concentration of
␣TP was calculated from the peak area responses using a standard
curve with ␣TP ranging from 110.5 to 442.3 pmol. The recovery of
the internal standard exceeded 94%.
Determination of 14C-OA absorption. From 100-␮L aliquots of
fresh lymph, 14C-radioactivity was determined with a liquid scintil-
lation counting system (Beckman LS-6500; Beckman Instruments)
after mixing with scintillation liquid (ScintiVerse; Fisher Scientific,
Fair Lawn, NJ). The 14C-radioactivity appearing in total hourly
lymph volumes was expressed as a percentage of the total 14C-
radioactivity infused.
Determination of lymphatic phospholipid (PL) and cholesterol
outputs. With 100 ␮L of hourly lymph samples, PL was measured
colorimetrically (UV-1201 Spectrophotometer; Shimazu Scientific
Instruments, Columbia, MD) according to the method of Raheja et
al. (18). Cholesterol was determined with the use of o-phthalalde-
hyde (Sigma Chemical), as described by Rudel and Morris (19).
Experiment 2
This experiment was conducted to determine whether the substi-
tution of lysoPC for PC in a lipid emulsion increases the intestinal
absorption of ␣TP and cholesterol. The protocols concerning diet
formulation, animal care, surgical procedure and lipid analyses were
the same as described for expt. 1, except that rats weighing 334 – 408
g were used for cannulation of the mesenteric lymph duct without
bile diversion. After an overnight recovery period, rats were infused
with a lipid emulsion containing 3.56 ␮mol ␣TP (all-rac-␣-tocoph-
erol, 97%; Aldrich Chemical), 33.3 kBq [4-14C]-cholesterol (specific
activity, 1.9 GBq/mmol; DuPont-New England Nuclear), 20.69 ␮mol
cholesterol, 452 ␮mol triolein (95%; Sigma Chemical) and 396 ␮mol
Na⫹-taurocholate with either 100 ␮mol DOPC (99%; Avanti Polar
Lipids) or lysoPC (99%; Avanti Polar Lipids) plus 100 ␮mol oleic
acid in 24 mL PBS (pH 6.6).
Statistical analysis. All statistical analyses were performed using
PC SAS (20). ANOVA and the least significant difference test were
performed to compare multiple group means and to detect time-
dependent changes within groups. Student’s t test was used to com-
pare two group means at designated time intervals. Differences were
considered significant at P ⬍ 0.05. Values in tables and figures are
expressed as means ⫾ SD.
 PHOSPHOLIPID AND VITAMIN E ABSORPTION 719

TABLE 2
Cumulative lymphatic absorptions of ␣-tocopherol (␣TP) and
14C-triolein (14C-OA) and outputs of phospholipid (PL) in bile-
diverted rats during duodenal infusion of a lipid emulsion with
1,2-dipalmitoyl phosphatidylcholine (DPPC) or 1,2-dilinoleoyl
phosphatidylcholine (DLPC) or without
phosphatidylcholine (NoPC)1

Lipid emulsion containing

NoPC DPPC DLPC

Lymph volume,

Downloaded from https://academic.oup.com/jn/article-abstract/131/3/717/4687210 by guest on 05 June 2020

FIGURE 1 The lymphatic absorption of ␣-tocopherol (␣TP) at mL/8 h 15.8 ⫾ 2.0a 16.7 ⫾ 4.2a 16.1 ⫾ 2.9a
hourly intervals for 8 h in bile-diverted rats during luminal infusion of a ␣TP
lipid emulsion without phosphatidylcholine (NoPC) or with either 1,2- nmol/8 h 692.2 ⫾ 135.0a 361.0 ⫾ 85.3b 383.8 ⫾ 63.4b
dipalmitoyl phosphatidylcholine (DPPC) or 1,2-dilinoleoyl phosphatidyl- % dose/8 h 13.9 ⫾ 2.7a 7.2 ⫾ 1.7b 8.5 ⫾ 1.4b
14C-OA,
choline (DLPC). All values are expressed as means ⫾ SD, n ⫽ 5.
*Significantly higher than DPPC and DLPC groups at the time point (P % dose/8 h 37.5 ⫾ 5.8b 53.1 ⫾ 7.4a 53.5 ⫾ 4.2a
⬍ 0.05). PL, ␮mol/8 h 21.2 ⫾ 4.2b 34.0 ⫾ 5.5a 32.4 ⫾ 2.4a

1 Means ⫾ SD, n ⫽ 5.
RESULTS Values in a row not sharing a superscript letter are different, (P
⬍ 0.05).
Experiment 1
Lymphatic absorption of ␣TP. The rates of lymph flow or
total lymph volumes did not differ regardless of whether PC Lymphatic output of PL. As expected, the enteral infu-
was infused. The hourly rates of lymph flow were 2.1 ⫾ 0.5 mL sion of PC markedly increased the hourly lymphatic outputs of
when infused with DPPC, 2.0 ⫾ 0.4 mL with DLPC and 2.0 PL. However, no difference (P ⬎ 0.05) was noted in PL
⫾ 0.2 mL with NoPC. Figure 1 shows the hourly lymphatic outputs between DPPC and DLPC rats (Fig. 3). After rapid
absorption of ␣TP in rats with bile diversion. The intraduo- increases for the initial 2 h, the rates of PL output in DPPC
denal infusion of either DPPC or DLPC in a lipid emulsion and DLPC rats remained constant at 4.0 –5.3 ␮mol/h. In rats
drastically lowered the hourly rates of ␣TP absorption at 4 h infused with no PC, the rates of PL output after 2 h ranged
and thereafter, with no significant difference between DPPC- from 2.6 to 3.0 ␮mol/h. The total amounts of PL released for
and DLPC-infused rats (Table 2). The hourly rates of ␣TP 8 h were 34.0 ⫾ 5.5 ␮mol in DPPC, 32.4 ⫾ 2.4 ␮mol in DLPC
absorption rose slowly but steadily in all groups during the first and 21.2 ⫾ 4.2 ␮mol in NoPC rats, with no significant
3 h of lipid infusion (Fig. 1). At 4 h and thereafter, however, difference between the PC groups (Table 2).
the hourly rates of absorption did not rise further in DPPC and
DLPC rats and remained at 50 to 60 nmol/h. In contrast, the Experiment 2
rate of ␣TP absorption in NoPC rats increased rapidly begin-
Lymphatic absorption of ␣TP and 14C-cholesterol (14C-
ning at 4 h and reached 122.8 ⫾ 23.4 nmol/h at 8 h. The rates
CH). Rates of lymph flow in lysoPC and DOPC rats were 2.6
of ␣TP absorption in DPPC, DLPC and NoPC groups were
⫾ 0.4 and 2.5 ⫾ 0.6 mL/h, respectively. The total volumes of
45.1 ⫾ 10.7, 48.0 ⫾ 7.9 and 86.5 ⫾ 16.9 nmol/h, respectively.
lymph secreted for 8 h were 20.5 ⫾ 3.5 mL in lysoPC rats and
The cumulative absorptions of ␣TP at 8 h were 361.0 ⫾ 85.3
nmol (7.2 ⫾ 1.7% dose) in DPPC, 383.8 ⫾ 63.4 nmol (8.5
⫾ 1.4% dose) in DLPC and 692.2 ⫾ 135.0 nmol (13.9 ⫾ 2.7%
dose) in NoPC rats, with no significant difference between the
DPPC and DLPC groups. The total absorptions of ␣TP in
DPPC and DLPC groups represented 52 and 56%, respec-
tively, of the controls (NoPC).
Lymphatic absorption of 14C-OA. In direct contrast to
the inhibitory effect of PC on ␣TP absorption, luminal infu-
sion of DPPC or DLPC markedly increased the absorption of
14
C-OA, as provided in the form of triolein. The hourly rates
of 14C-OA absorption rose rapidly in rats infused with DPPC
or DLPC starting at 3 h and continued to rise above the rates
observed in NoPC rats until 8 h (Fig. 2). The rates of 14C-OA
absorption were 6.6 ⫾ 0.9% dose/h in DPPC, 6.7 ⫾ 0.5%
dose/h in DLPC and 4.7 ⫾ 0.6% dose/h in NoPC rats. The
hourly rates of 14C-OA absorption in both DPPC and DLPC
FIGURE 2 The lymphatic absorption of triolein labeled with 14C
rats steadily increased in close parallel, with no significant (14C-OA) at hourly intervals for 8 h in bile-diverted rats during luminal
difference at any hourly intervals (Fig. 2). The cumulative infusion of a lipid emulsion without phosphatidylcholine (NoPC) or with
absorptions of 14C-OA for 8 h in DPPC and DLPC rats were either 1,2-dipalmitoyl phosphatidylcholine (DPPC) or 1,2-dilinoleoyl
53.1 ⫾ 7.4% dose and 53.5 ⫾ 4.2% dose, respectively, which phosphatidylcholine (DLPC). All values are expressed as means ⫾ SD,
were significantly higher than in NoPC rats (37.5 ⫾ 5.8% n ⫽ 5. *Significantly lower than DPPC and DLPC at the time point (P
dose). ⬍ 0.05).
720 KOO AND NOH

Downloaded from https://academic.oup.com/jn/article-abstract/131/3/717/4687210 by guest on 05 June 2020

FIGURE 3 The lymphatic output of phospholipid (PL) at hourly
intervals for 8 h in bile-diverted rats during luminal infusion of a lipid
emulsion without phosphatidylcholine (NoPC) or with either 1,2-di-
palmitoyl phosphatidylcholine (DPPC) or 1,2-dilinoleoyl phosphatidyl-
choline (DLPC). All values are expressed as means ⫾ SD, n ⫽ 5.
*Significantly lower than DPPC and DLPC at the time point (P ⬍ 0.05).

20.1 ⫾ 5.1 mL in DOPC rats, with no significant difference

between groups. However, the luminal infusion of lysoPC
significantly increased the lymphatic absorption of ␣TP com-
pared with that of DOPC (Fig. 4A, P ⬍ 0.05). The hourly rate
of ␣TP absorption was increased sharply with lysoPC infusion,
peaking at 78.9 ⫾ 17.6 nmol/h at 3 h, and then declined to a
rate of 73.0 nmol/h thereafter. In rats infused with DOPC,
however, the absorption of ␣TP, after peaking at 69.5 ⫾ 20.7
nmol/h at 3 h, decreased to 53.0 ⫾ 12.2 nmol/h at 4 h and
remained at that level. The rates of ␣TP absorption were 73.0
⫾ 8.3 nmol/h in lysoPC rats and 51.1 ⫾ 8.3 nmol/h in DOPC
rats. The cumulative absorptions of ␣TP for 8 h were 583.7
⫾ 12.8 nmol (16.4 ⫾ 0.4% dose) in lysoPC rats and 408.5
⫾ 66.5 nmol (11.5 ⫾ 1.9% dose) in DOPC rats (Table 3).
Similarly, the lymphatic absorption of 14C-CH was signifi-
cantly higher in lysoPC rats than in DOPC rats at 3 h and
thereafter (Fig. 4B, P ⬍ 0.05). The hourly rates of 14C-CH
absorption were 4.2 ⫾ 0.1% dose/h in lysoPC rats and 3.1
⫾ 0.3% dose/h in DOPC rats. The cumulative absorption of
14
C-CH for 8 h also was significantly higher in lysoPC rates
(33.7 ⫾ 1.1% dose) than in DOPC rats (24.8 ⫾ 2.1% dose)
(Table 3).
Lymphatic output of PL. Rates of PL secretion in rats
infused with lysoPC and DOPC were 3.6 ⫾ 0.3 and 3.2 ⫾ 0.4
␮mol/h, respectively, with no significant difference between
groups. The cumulative lymphatic outputs of PL in lysoPC and
DOPC rats for 8 h were 29.1 ⫾ 2.5 and 25.8 ⫾ 3.4 ␮mol,
respectively, with no significant difference between groups.
DISCUSSION
In the present study using bile-diverted rats with lymph
cannulas, we provided clear evidence that the inclusion of
intact PC in a lipid emulsion causes drastic decreases in the
lymphatic absorption of ␣TP and cholesterol, whereas it en-
hances the absorption of fat (or fatty acid). In addition, the
present data demonstrate that the substitution of lysoPC for
PC simultaneously increases the absorption of ␣TP and cho-
lesterol. Data show that the inhibitory effects of PC on ␣TP
and cholesterol absorption are unrelated to the chain length or
saturation/unsaturation of the acyl groups of the PC infused.
To compare the effects of exogenous PC (DPPC versus
DLPC), the influence of biliary PC was eliminated by bile
FIGURE 4 The lymphatic absorptions of ␣-tocopherol (␣TP) (A)
and 14C-cholesterol (14C-CH) (B) at hourly intervals for 8 h in rats during
luminal infusion of a lipid emulsion with either 1-oleoyl-2-hydroxy phos-
phatidylcholine (lysoPC) or 1,2-dioleoyl phosphatidylcholine (DOPC).
All values are expressed as means ⫾ SD, n ⫽ 5. *Significant differences
between groups at the time point (P ⬍ 0.05).
diversion, but Na⫹-taurocholate was provided at 49.5 ␮mol/h
to ensure an adequate supply of bile salts (21).
At the present, the mechanism underlying such dual effects
of PC on lipid absorption is far from clear. Although intact PC
is taken up via the intestinal brush border membrane (22), its
direct uptake is thought to be minimal (23). Considerable
evidence indicates that the action of intact PC is largely
intraluminal. Studies, as mostly conducted under in vitro
TABLE 3
Cumulative lymphatic absorptions of ␣-tocopherol (␣TP) and
14C-cholesterol (14C-CH) and output of phospholipid (PL) in
rats with lymph cannula during duodenal infusion of a lipid
emulsion containing either 1-oleoyl-2-hydroxy
phosphatidylcholine (lysoPC) or 1,2-dioleoyl
phosphatidylcholine (DOPC)1
DOPC LysoPC
␣TP
nmol/8 h 408.5 ⫾ 66.5 583.7 ⫾ 12.8*
% dose/8 h 11.5 ⫾ 1.9 16.4 ⫾ 0.4*
14C-CH, % dose/8 h 24.8 ⫾ 2.1 33.7 ⫾ 1.1*
PL, ␮mol/8 h 25.8 ⫾ 3.4 29.1 ⫾ 2.5
1 Means ⫾ SD, n ⫽ 5.
* Significantly different from DOPC (P ⬍ 0.05).
 PHOSPHOLIPID AND VITAMIN E ABSORPTION
conditions, suggest that PC may affect the intestinal absorp-
tion of lipids by influencing the following intraluminal events:
1) the rates of lipolysis, 2) formation and diffusion of mixed
micelles across the unstirred water layer and 3) transfer of
micellar lipids to the brush border membrane. Earlier studies
have shown that the presence of PC on the surface of lipid
emulsions hinders the hydrolysis of the core TG by pancreatic
lipase even in the presence of bile salts and colipase, whereas
a limited initial hydrolysis of PC to lysoPC by pancreatic PLA2
facilitates the binding of lipase/colipase to the substrate inter-
face, resulting in a rapid hydrolysis of TG to fatty acids and
monoacylglycerol (24 –26). More recently, a study using rat
IEC-6 intestinal cells (12) also showed that pancreatic lipase/
colipase was ineffective in hydrolyzing TG in PC-containing
lipid emulsions and that the initial hydrolysis of the surface PC
by the addition of pancreatic PLA2 significantly increased the
ability of pancreatic lipase/colipase to hydrolyze the core TG
of the emulsion. Minimal hydrolysis of TG was required for
stimulation of the cell uptake of cholesterol, suggesting that
fatty acid and monoacylglycerol liberated from TG are key
determinants in facilitating cholesterol transfer to the entero-
cyte. Thus, the combined actions of pancreatic PLA2 and
lipase/colipase are critical for support of the normal rates of
luminal lipolysis and subsequent formation of mixed micelles.
The inclusion of PC in mixed micelles also has been shown to
increase the solubility of lipids and the size of micelles in the
presence of bile salts, which results in slowing of the rate of
micellar diffusion across the unstirred water layer (6,8,27).
Thus, these observations suggest that before its hydrolysis to
lysoPC, PC may affect the intestinal uptake of lipids by slow-
ing the rates of lipolysis and micelle formation in the intestinal
lumen.
An important observation from the present study is that
exogenous PC in bile-diverted rats inhibits the absorption of
␣TP and cholesterol, whereas it enhances the absorption of fat
under in vivo conditions. It should be pointed out that the
amount of PC infused was more than adequate to provide the
amount of biliary PC lost by bile diversion, which was deter-
mined to be 3– 4 ␮mol/h (unpublished data). In the present
study, the amount of PC infused in the bile-diverted rats was
set at 10 ␮mol/h to provide the amount of PC lost (4.0 ␮mol)
by bile diversion and the estimated intake of PC through a
typical diet. On a daily basis, this amount is equal to an intake
of 113 mg PC and equivalent to 0.38 mg/kJ based on the
average daily food intake of 20 g (AIN-93G diet), providing
297 kJ. For a human who consumes 10,450 kJ (2500 kcal)/d, it
would be equivalent to a daily intake of 4.0 g PC, which is
within the range (4 – 8 g) of daily PC intake estimated for
adult humans (28). Thus, the observed inhibitory effect of PC
on ␣TP and cholesterol absorption is not attributable to a lack
or excess of PC supply. The inhibition of ␣TP absorption by
PC also was demonstrated previously by using the rat small
intestine perfused in situ with egg PC incorporated into bile
salt micelles (13). However, the mechanism underlying the
dual effects of PC (i.e., inhibition of ␣TP and cholesterol
absorption and enhancement of fat absorption) has yet to be
understood. It is generally believed that ␣TP and other fat-
soluble vitamins enter the intestinal cell via passive diffusion,
moving along with absorbed lipids (29 –31). However, a recent
in vitro study using Caco-2 cells (10) demonstrated that the
presence of intact PC in bile salt micelles differentially affects
the cell uptake of lipids from the micellar matrix. The data
showed that PC markedly reduced the uptake of cholesterol by
Caco-2 cells, whereas it did not interfere with the cell uptake
of oleic acid and monoacylglycerol (10). The investigators
postulated that the presence of intact PC in bile salt micelles
721
slows the transfer (desorption) of more hydrophobic lipids
such as cholesterol (a 27-carbon lipid), without affecting the
transfer of other less hydrophobic lipids, such as fatty acid and
monoacylglycerol, products of TG hydrolysis by pancreatic
lipase/colipase. This hypothesis is supported by the earlier
observation by Pownall et al. (32) that the rate of transfer of
a hydrophobic molecule from PC single bilayer vesicles de-
creases with increasing hydrophobicity of the molecule. Con-
sistent with these observations is the present finding that
luminally infused PC slows the lymphatic absorption of ␣TP,
an extremely hydrophobic 29-carbon lipid, whereas it en-
hances the absorption of fat (oleic acid).
The present data clearly demonstrate that the inhibitory
effect of PC on the absorption of ␣TP and cholesterol is
Downloaded from https://academic.oup.com/jn/article-abstract/131/3/717/4687210 by guest on 05 June 2020
abolished when lysoPC is substituted for PC. Several studies
previously have shown that the inclusion of lysoPC in mixed
micelles or addition of pancreatic PLA2 in mixed micelles or a
lipid emulsion enhances cholesterol uptake by intestinal cells
under in vitro conditions (10 –12). The present study is the
first to show under in vivo conditions that the substitution of
lysoPC for PC in a lipid emulsion simultaneously enhances the
absorption of ␣TP and cholesterol. This effect of lysoPC may
be mediated not only by its favorable effect on TG hydrolysis
and micellar formation in the intestinal lumen but also by its
stimulation of intracellular processing of lipids within the
enterocyte. It is well documented that once lysoPC is taken up
by the enterocyte, it is reconverted to PC and facilitates the
intracellular reacylation and packaging of lipids and the for-
mation and secretion of chylomicrons (1,21,33–36).
In summary, the present study provides evidence that the
luminal infusion of PC in bile-diverted rats with lymph can-
nulas drastically lowers the absorption of ␣TP but markedly
increases the lymphatic absorption of fat and the output of PL.
The substitution of lysoPC for PC in a lipid emulsion signifi-
cantly enhances the intestinal absorption of ␣TP and choles-
terol. Thus, pancreatic PLA2, which hydrolyzes PC to lysoPC,
may play an important role in regulating the absorption of
lipids and lipid-soluble vitamins. Our data here, as obtained
under in vivo conditions, suggest that exogenous or dietary PC
may be used to lower cholesterol absorption but may adversely
affect the body or nutritional status of vitamin E by decreasing
its absorption. Further investigation is required to delineate
the impacts of dietary PC intake on ␣TP status and cholesterol
metabolism.
LITERATURE CITED
1. O’Doherty, P. J., Kasis, G. & Kuksis, A. (1973) Role of luminal lecithin
in intestinal fat absorption. Lipids 8: 249 –255.
2. Tso, P. (1994) Intestinal lipid absorption. In: Physiology of the Gastro-
intestinal Tract (Johnson, L. R., Alpers, D. H., Christensen, J., Jacobson, E. D. &
Walsh, J. H., eds.), 3rd ed., pp. 1867–1907, Raven Press, New York, NY.
3. Tso, P. & Scobey, M. (1986) The role of phosphatidylcholine in the
absorption and transport of dietary fat. In: Fat Absorption (Kuksis, A., ed.), vol. 1,
pp. 177–195. CRC Press, Boca Raton, FL.
4. Hollander, D. & Morgan, D. (1980) Effect of plant sterols, fatty acids
and lecithin on cholesterol absorption in vivo in the rat. Lipids 15: 395– 400.
5. Rampone, A. J. (1973) Effect of lecithin on intestinal cholesterol up-
take by rat intestine in vitro. J. Physiol. 229: 505–514.
6. Rampone, A. J. & Long, L. W. (1977) The effect of phosphatidylcholine
and lysophosphatidylcholine on the absorption and mucosal metabolism of oleic
acid and cholesterol in vitro. Biochim. Biophys. Acta 486: 500 –510.
7. Reynier, M. O., Lafont, H., Crotte, C., Sauve, P. & Gerolami, A. (1985)
Intestinal cholesterol uptake: comparison between mixed micelles containing
lecithin or lysolecithin. Lipids 20: 145–150.
8. Thomson, A. B. & Cleland, L. (1981) Intestinal cholesterol uptake from
phospholipid vesicles and from simple and mixed micelles. Lipids 16: 881– 887.
9. Beil, F. U. & Grundy, S. M. (1980) Studies on plasma lipoproteins
during absorption of exogenous lecithin in man. J. Lipid Res. 21: 525–536.
10. Homan, R. & Hamelehle, K. L. (1998) Phospholipase A2 relieves phos-
phatidylcholine inhibition of micellar cholesterol absorption and transport by
human intestinal cell line Caco-2. J. Lipid Res. 39: 1197–1209.
722 KOO AND NOH
11. Mackay, K., Starr, J. R., Lawn, R. M. & Ellsworth, J. L. (1997) Phos-
phatidylcholine hydrolysis is required for pancreatic cholesterol esterase- and
phospholipase A2-facilitated cholesterol uptake into intestinal Caco-2 cells.
J. Biol. Chem. 272: 13380 –13389.
12. Young, S. C. & Hui, D. (1999) Pancreatic lipase/colipase-mediated
triacylglycerol hydrolysis is required for cholesterol transport from lipid emulsions
to intestinal cells. Biochem. J. 339: 615– 620.
13. Imai, J., Hayashi, M., Awazu, S. & Hanano, M. (1983) Intestinal ab-
sorption of dl-␣-tocopherol from bile salts and polysorbate 80 micellar solutions
in rat. J. Pharm. Dyn. 6: 897–902.
14. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr. (1993) AIN-93 purified
diets for laboratory rodents: final report of the American Institute of Nutrition Ad
Hoc Writing Committee on the reformulation of the AIN-76A rodent diet. J. Nutr.
123: 1939 –1951.
15. Noh, S. K. & Koo, S. I. (1997) The lymphatic absorption of lipids is
normalized by enteral phosphatidylcholine infusion in ovariectomized rats with
estrogen replacement. J. Nutr. Biochem. 8: 152–161.
16. Noh, S. K., Koo, S. I. & Jeon, I. J. (1999) Estradiol replacement in
ovariectomized rats increases the hepatic concentration and biliary secretion of
␣-tocopherol and polyunsaturated fatty acids. J. Nutr. Biochem. 10: 110 –117.
17. Zaspel, B. J. & Csallany, A. S. (1983) Determination of alpha-toco-
pherol in tissues and plasma by high-performance liquid chromatography. Anal.
Biochem. 130: 146 –150.
18. Raheja, R. K., Kaur, C., Singh, A. & Bhatia, I. S. (1973) New colori-
metric method for the quantitative estimation of phospholipids without acid
digestion. J. Lipid Res. 14: 695– 697.
19. Rudel, L. L. & Morris, M. D. (1973) Determination of cholesterol using
o-phthalaldehyde. J. Lipid Res. 14: 364 –366.
20. SAS Institute, Inc. (1985) SAS User’s Guide: Statistics, Version 5, pp.
433–506. SAS Institute, Cary, NC.
21. Tso, P., Lam, J. & Simmonds, W. J. (1978) The importance of the
lysophosphatidylcholine and choline moiety of bile phosphatidylcholine in lym-
phatic transport of fat. Biochim. Biophys. Acta 528: 364 –372.
22. Thurnhofer, H. & Hauser, H. (1990) The uptake of phosphatidylcholine
by small intestinal brush border membrane is protein-mediated. Biochim. Bio-
phys. Acta 1024: 249 –262.
23. Parthasarathy, S., Subbaiah, P. V. & Ganguly, J. (1974) The mecha-
nism of intestinal absorption of phosphatidylcholine in rats. Biochem. J. 140:
503–508.
24. Blackberg, L., Hernell, O. & Olivecrona, T. (1981) Hydrolysis of human
milk fat globules by pancreatic lipase: role of colipase, phospholipase A2, and bile
salts. J. Clin. Invest. 67: 1748 –1752.
25. Borgström, B. (1980) Importance of phospholipids, pancreatic phos-
pholipase A2, and fatty acid for the digestion of dietary fat: in vitro experiments
with the porcine enzymes. Gastroenterology 78: 954 –962.
26. Patton, J. S. & Carey, M. C. (1981) Inhibition of human pancreatic
lipase– colipase activity by mixed bile salt-phospholipid micelles. Am. J. Physiol.
241: G328 –G336.
27. Rampone, A. J. & Machida, C. M. (1981) Mode of action of lecithin in
suppressing cholesterol absorption. J. Lipid Res. 22: 744 –752.
28. Carey, M. C., Small, D. M. & Bliss, C. M. (1983) Lipid digestion and
absorption. Annu. Rev. Physiol. 45: 651– 677.
29. Hollander, D. (1981) Intestinal absorption of vitamins A, E, D, and K. J.
Lab. Clin. Med. 97: 449 – 462.
30. Kayden, H. J. & Traber, M. G. (1993) Absorption, lipoprotein transport,
Downloaded from https://academic.oup.com/jn/article-abstract/131/3/717/4687210 by guest on 05 June 2020
and regulation of plasma concentrations of vitamin E in humans. J. Lipid Res. 34:
343–358.
31. Muralidhara, K. S. & Hollander, D. (1977) Intestinal absorption of
␣-tocopherol in the unanesthetized rats: the influence of luminal constituents on
the absorptive process. J. Lab. Clin. Med. 90: 85–91.
32. Pownall, H. J., Hickson, D. L. & Smith, L. C. (1983) Transport of
biological lipophiles: effect of lipophile structure. J. Am. Chem. Soc. 105: 2440 –
2445.
33. Clark, S. B. (1978) Chylomicron composition during duodenal triglyc-
eride and lecithin infusion. Am J. Physiol. 235: E183–E190.
34. Mansbach, C. M. II, Arnold, A. & Cox, M. A. (1985) Factors influencing
triacylglycerol delivery into mesenteric lymph. Am. J. Physiol. 249: G642–G648.
35. O’Doherty, P. J., Yousef, I. M. & Kuksis, A. (1974) Effect of phosphati-
dylcholine on triacylglycerol synthesis in rat intestinal mucosa. Can. J. Biochem.
52: 726 –733.
36. Tso, P., Kendrick, H., Balint, J. A. & Simmonds, W. J. (1981) Role of
biliary phosphatidylcholine in the absorption and transport of dietary triolein in the
rat. Gastroenterology 80: 60 – 65.