The effect of phosphatidylcholine and lysophosphatidylcholine on the absorption and mucosal metabolism of oleic acid and cholesterol in vitro

500

Biochimica et Biophysics @ Elsevier/North-Holland

Acta, 486 (1977) Biomedical Press

590-510

BBA 56951

THE EFFECT OF PHOSPHATIDYLCHOLINE AND LYSOPHOSPHATIDYLCHOLINE ON THE ABSORPTION AND MUCOSAL METABOLISM OF OLEIC ACID AND CHOLESTEROL IN VITRO

ALFRED

J. RAMPONE

and LAWRENCE

R. LONG

Department of Physiology, School of Medicine, Center, Portland, Oreg. 97201 (U.S.A.) (Received

August

30th,

Ilniuersity

of Oregon

Health

Sciences

1976)

Summary

The absorption and mucosal metabolism of [ 14C]oleic acid and [ 3H]cholesterol were studied using everted sacs of rat jejunum in an in vitro incubation system. The labeled compounds were present in the incubation mixture either singly or together as mixed micelles with bile salt and monoacylglycerol and in the presence or absence of phosphatidylcholine or lysophosphatidylcholine. The presence of cholesterol or phosphatidylcholine markedly suppressed oleic acid absorption. We suggest that both compounds interacted with the micelles causing changes in micellar mass, charge or configuration leading to possible interference with access of the fatty acid to the cell membrane. Lysophosphatidylcholine enhanced oleic acid absorption and stimulated incorporation of the fatty into mucosal triacylglycerol. When the incubation temperature was lowered to suppress metabolism lysophosphatidylcholine had no effect. The results suggest that the increased absorption occurring at the higher temperature was secondary to enhanced glycerol acylation. Lysophosphatidylcholine had only a minimal effect on cholesterol absorption and no effect on cholesterol acylation. Evidence is presented showing that lysophosphatidylcholine is itself well absorbed and variously metabolized. We conclude that phosphatidylcholine and lysophosphatidylcholine have quite divergent effects on lipid absorption but the full elucidation of their mechanisms of action must await further study.

Introduction

Phosphatidylcholine has been shown to suppress micellar fatty acid and cholesterol uptake by rat intestine in vitro [ 1,2]. It was suggested in these studies that the effect was possibly due to the amphipathic properties of phos-

501

phatidylcholine causing it to incorporate into and increase the size of the mixed lipid-bile acid micelles thus reducing the coefficient of free diffusion of the micelles. Recent experiments have shown that an unstirred water layer overlying the epithelial surface of the intestine represents an important barrier to lipid uptake, diffusion through the layer being rate-limiting according to Dietschy and co-workers [3,4]. This important concept explains how micelle formation improves the efficiency of lipid absorption since by transporting lipid molecules in the form of polar aggregates the micelle overcomes some of the disadvantage associated with the low aqueous solubility of fatty acid and cholesterol monomers. The micelle, therefore, helps to establish contact between the lipids and the cell membrane by bridging the “moat” or unstirred water layer. The advantage afforded by the micellar aggregates in transporting the lipids across the unstirred water layer and improving access of the lipids to the cell membrane is dependent to some extent on the coefficient of free diffusion of the micelles in the unstirred water layer, and this in turn is dependent on micellar mass and mobility. Some of the advantages of, micelle formation, therefore, may be offset by the presence of agents, such as phosphatidylcholine, which cause micelles to swell and increase in size [ 5,6]. This idea provides a possible explanation for the previously discovered suppressor effect of phosphatidylcholine on micellar fatty acid and cholesterol uptake in vitro

[1,21. The situation may be different in vivo since phosphatidylcholine is readily split in the lumen to lysophosphatidylcholine by the action of pancreatic phospholipase A enzyme [7,8]. The question then arises as to whether or not lysophosphatidylcholine has any effect on fatty acid and cholesterol uptake under conditions similar to those in which the suppressor effects of phosphatidylcholine were demonstrated. The present study was directed to that end. We found that the lyso derivative contrasted sharply with phosphatidylcholine in causing a marked enhancement of fatty acid uptake with only a minimal effect on cholesterol uptake. Further experiments were then done to determine if the enhanced fatty acid uptake was a primary effect of lysophosphatidylcholine on membrane permeation or a secondary effect due to a possible stimulation of fatty acid incorporation into tissue triacylglycerols. Materials and Methods Chemicals

Phosphatidylcholine was extracted and purified from rat livers as previously described [ 21. L-cY-Lysophosphatidylcholine (98%) was purchased from Sigma Chemical Company or made enzymatically from the biosynthesized phosphatidylcholine by incubating the purified compound in the presence of fresh pancreatic juice obtained from donor rats. The pancreatic juice was preheated for 10 min at 60” C to inactive lipases other than phospholipase A [ 91. Incubations were carried out according to the method of Hanahan [lo] as modified by Long and Penny [ 111. The procedure cleaves the 2-ester linkage leaving the 1-acyl lysophosphatidylcholine [9]. The isolated compound was purified on

502

columns of silicic acid [7] and the purity verified by thin-layer chromatography [12]. [ ‘H] Glycerol-labeled lysophosphatidylcholine and [ .‘H] -palmitate-labeled lysophosphatidylcholine were sirnilarly prepared and purified from rat livers 6 h after the intraperitoneal injection of 5 mCi of [ 3H]glycerol or [ “Hlpalmitate respectively (New England Nuclear). Both chemicals had radiochemical purities of >98%. The palmitate was complexed to 4% bovine serum albumin for purposes of injection [ 131. Sources of other chemicals were as follows: oleic acid (99%) and sodium taurocholate (reag.), ICN Pharmaceuticals; monoacylglycerol (90%), Cal Biothem; cholesterol (99%), Sigma Chemical Co.; [l-‘4C]oleic acid and [1,2‘H]cholesterol, New England Nuclear. The radioactive chemicals had stated radiochemical purities of >98% and were used as supplied. Experimental procedures The experiments utilized unfasted male Sprague-Dawley rats weighing 250300 g. The details of the preparation of everted intestinal jejunal sacs and the micellar incubation solutions have been described previously [ 141. The micellar solutions were optically clear and contained oleic acid 0.6 mM, monoacylglycerol 0.3 mM, cholesterol (when present) 0.15 mM, sodium taurocholate 4.8 mM, and glucose 20 mM made up in phosphate buffer, pH 6.5 without calcium or magnesium. When oleic acid and cholesterol uptakes were measured these compounds were labeled with trace amounts of [l-‘4C]oleic acid and [ 1 ,2-3H]cholesterol respectively. When the effects of phosphatidylcholine or lysophosphatidylcholine were assessed these compounds were added to the micellar solutions in concentrations ranging from 0.1 to 2.4 mM based on the palmitoyl forms. When lysophosphatidylcholine uptake was measured it was labeled with “H in either the palmitate or the glycerol moiety and the oleic acid and cholesterol were unlabeled. Except where indicated all incubations were for one hour in an oxygenated atmosphere at 37OC with constant agitation of the flasks at a controlled rate of 80 oscillations/min. Following incubation the sacs were removed and washed thorougly by swirling in cold saline. The adherent wash fluid was then removed with absorbent paper and the serosal fluid, which consisted initially of 2 ml of phosphate buffer alone, was discarded (the results of preliminary experiments indicated that the serosal fluid gained only negligible quantities of radioactivity). The empty sacs were transferred to chilled stainless steel plates and the mucosa was scraped off with a spatula, transferred to a large centrifuge tube and weighed to obtain tissue wet weight. Lipid extractions and separation into various lipid fractions were carried out as previously described [ 14,151. A three channel liquid scintillation spectrometer system (Packard Instrument Co.) was used to monitor radioactivity using the Automatic External Standard to correct for quenching and spillover of 14C into the “H channel. There was no spillover of 3H into the 14C channel. One ml of the original incubation fluid was extracted and counted along with the tissue samples and served as a standard of known specific activity to convert dpm to mass. Uptakes were expressed as nmol/g tissue wet weight/hour. In a few experiments the incubation temperature was lowered to near O°C

503

to suppress metabolism. The flasks were immersed in a salt-ice water slush to maintain the incubation fluid at 0-4°C. All procedures following incubation were carried out in a cold room. Results Oleic acid uptake experiments [‘4C]Oleic acid uptake was measured in single isotope experiments and in double isotope experiments in which [ “C]oleic acid and [ 3H]cholesterol were present together. Uptakes were measured in the absence of and in the presence of varying concentrations of phosphatidyl~holine or lysophosphatidyl~holine. Fig. 1 shows the effects of phosphatidylcholine on oleic acid uptake and its incorporation into (a) mucosal total lipid (T) measured in an aliquot of the chloroform/methanol extract before chromatography and (b) mucosal triacylglycerol (TG) and free fatty acid (FFA) measured after thin-layer chromatography. In these first experiments cholesterol was omitted from the incubation

rT

Phosphatldylchollne

Concentration

t mM)

Phosphat~dylcholme

Concentratmn

( m Ml

Fig. 1. Effect of varying concentrations of phosphatidylcholine on mice&r 1’ 4C]oleic acid uptake and incorporation into mucosal triacylglycerol in everted jejunal sacs. Oleic acid concentration in the incubation medium was 0.6 mM. monoacylglycerol concentration was 0.3 mM and sodium taurocholate concentration was 4.8 mM. Recoveries of ‘*C in three lipid fractions are shown: total lipid (T). triacylglycerol (TG) and free fatty acid (FFA). Vertical bars are standard errors of the means in 6 to 10 experiments (N). Fig. 2. Effect of varying concentrations of phosphatidylcholine on mieellar [’ 4C]oleic acid uptake and incorporation into mucosal triacylglycerol in everted jejunal sacs in the presence of cholesterol. Experiments were the same as those described in Fig. 1 except that cholesterol was present in a concentration of 0.15 mM. Symbols as in Fig. 1.

504

medium. In confirmation of an earlier study by Rodgers and O’Connor [ 11, phosphatidylcholine was found to have a marked inhibitory effect on oleic acid uptake, maximum inhibition occurring at a concentration of approx. 0.5 mM and showing evidence of saturation at this and higher concentrations. The tissue lipid fractions containing the major portion of the radioactivity were triacyglycerol and free fatty acid (Fig. 1). In the control experiments (zero phosphatidylcholine concentration) the radioactivity was present mostly in the free fatty acid, whereas in the presence of phosphatidylcholine it was mostly in triacylglycerol. Fig. 2 shows the effect of phosphatidylcholine on oleic acid uptake in the presence of cholesterol (0.15 mM). The curves are comparable to those in Fig. 1 except in the control series on the left. This difference from the previous controls (Fig. 1) suggests that cholesterol alone was sufficient to suppress oleic acid uptake. The addition of phosphatidylcholine produced a further suppression which was comparable to that seen in Fig. 1. In all of the experiments, possibly including the controls in this case, the major portion of the radioactivity was in triacylglycerol. The effect of lysophosphatidylcholine on oleic acid uptake and its incorporation into tissue lipids was studied under similar conditions but only in the presence of cholesterol (0.15 mM) so that results are best compared with those in Fig. 2. It will be observed (Fig. 3) that this phospholipid induced changes which were, in some respects, opposite to those induced by phosphatidylcholine. Thus, there was a marked stimulation of 14C uptake (T) rather than an inhibition, most of the increase being accounted for as triacylglycerol (TG). Maximum stimulation occurred at a concentration near 0.8 mM with apparent saturation occurring at higher concentrations. The amount of label recovered in the tissue free fatty acid (FFA) actually decreased in the presence of the compound despite the marked increase in total uptake. Presumably lysophosphatidylcholine had a major influence in enhancing acylation of the absorbed fatty acid to triacylglycerol. Triacylglycerol then accumulated in the tissue because, as mentioned before, transport of lipids into the serosal fluid is negligible under these in vitro conditions. Only small amounts of the label appeared in the tissue phospholipids in Fig. 3, the amount increasing slightly in the presence of lysophosphatidylcholine. Further support for the idea that lysophosphatidylcholine stimulates esterification of fatty acid was obtained in additional experiments in which the incubation temperature was lowered to near 0°C. Processes, such as esterification, having a high temperature coefficient will be greatly suppressed at 0°C [ 161 compared to processes having a low temperature coefficient, such as fatty acid uptake, which has been shown in other studies to be a passive process [17, 181. The results of the 0°C incubation experiments are shown in Table I. In the controls (absence of lysophosphatidylcholine) the total uptake (T) at 0°C was comparable to that at 37’C (Fig. 3). Most of the label, as expected, was in the free fatty acid form and significantly less in the triacylglycerol form, contrasting sharply with the 37OC experiments. Only small amounts of label appeared in the tissue phospholipids. The addition of lysophosphatidylcholine in a concentration of 0.8 mM at

,505

1i I ,/‘I /’

_-- -----

__--‘4

/’

1

;;)p

LysophosphotldylcholIne

i-L+ I

I

Concentroi~on

( m M 1

Lysophosphotidylcholme

Concentrot~on

( mM1

Fig. 3. Effect of varying concentrations of lysophosphatidylcholine on micelIar [’ 4Cloleic acid uptake and incorporation into mucosal lipids in everted jejunal sacs. Experiments were the same as those described in Fig. 2 except that lysophosphatidylcholine replaced phosphatidylcholine. Recoveries in four lipid fractions are shown: total lipid (T). triacylglycerol (TG), free fatty acid (FFA) and phospholipid (PL). Vertical bars are standard errors of means of 4 to 13 experiments (N). Fig. and terol Fig.

4. Effect of varying concentrations of lysophosphatidylcholine on micellar [ ‘HIcholesterol uptake incorporation into mucosal cholesterol ester in everted jejunal sacs. Recovery of .‘H in total choles(TC) and free cholesterol (FC) are shown. The sacs were the same ones used to obtain the data in 3. Vertical bars are standard errors of means in 4 to 13 experiments (N).

0°C failed to stimulate uptake or alter the pattern of distribution of the label in the different lipid fractions, again contrasting sharply with the earlier experiments of Fig. 3. Since uptake was not enhanced, the data suggest that the previously observed stimulating effect of the compound on uptake was probably secondary to an increased rate of esterification.

TABLE

I

f14C10LEIC ACID UPTAKE AND INCORPORATION AND ABSENCE OF LYSOPHOSPHATIDYLCHOLINE BATED AT NEAR O°C

INTO MUCOSAL LIPIDS IN THE PRESENCE (LPC) IN EVERTED JEJUNAL SACS INCU-

The incubation fluid contained oleic acid, 0.6 mM: monoacylglycerol, 0.3 mM; sodium taurocholate, 4.8 mM: cholesterol, 0.15 mM. Recoveries of 14C in four lipid fractions are shown: total lipid (T), triacylglycerol (TG). free fatty acid (FFA) and phospholipid (PL). Values are means f standard error of means in eight experiments at each LPC concentration. LPC concentration

0

0.8 mM

-

14C recovery in mucosal lipids (nmol/g tissue wet weight/h)

~_______

T

TG

FFA

PL

1246 + 86 1231 + 104

211 + 32 285 f 27

829 + 48 174 * IO

37 + 1.8 49 & 4.8

____.___

506

Uptake of labeled lysophos~hat~dy~chol~ne The argument that lysophosphatidylcholine has an intracellular metabolic effect, as suggested in the above experiments, would be strengthened if it could be shown that the lysophosphatidylcholine itself is absorbed under these in vitro conditions. Accordingly, its absorption was measured in two separate sets of experiments at 37°C using lysophosphatidylcholine labeled with 3H in either the glycerol or palmitate moiety. The experiments were as described in the preceding section except that only the lysophosphatidylcholine was labeled and the labels on fatty acid and cholesterol were omitted. The distribution of the label in the tissue lipid fractions after incubation was similarly determined. The Iysophosphatidylcholine concentration was again 0.8 mM considered to be near maximum in terms of its previously demonstrated stimulating effects. The results are summarized in Table II. They show that significantly more of the label was recovered in the total lipid (T) and triacylglycerol (TG) when the palmitate was labeled than when the glycerol was labeled suggesting that some of the material was hydrolyzed with loss of some of the glycerol. A considerable amount (1178 nmol or 19%) of the absorbed palmitate was unaccounted for, for unknown reasons. The amount of label incorporated into the tissue phospholipids (PL) was uniform and independent of the position of the label. The amounts of labeled phospholipid in both cases, however, were significantly greater than in the previous experiments utilizing labeled oleic acid with or without added lysophosphatidylcholine (Fig. 3). Cholesterol uptake experiments Cholesterol uptake was shown in earlier studies of this type to be markedly suppressed in the presence of phosphatidylcholine 121, the results having been confirmed by Rodgers and O’Connor [l]. In the present study the effects of lysophosphatidylcholine were tested under similar conditions and the results are shown in Fig. 4. There was an apparent slight stimulation of cholesterol uptake by lysophosphatidylchol~ne at the lower concentrations but the increase was not sustained at the higher concentration. Only the increases observed at concentrations of 0.8 and 1.2 mM were significant compared to control values without lysophosphatidylcholine (0.01 < P < 0.02). The stimulation was much

TABLE

II

UPTAKE INTO LPC

was

It was (0.3

OF

LABELER

.MUCOSAL present

dissolved

mM),

of means Labeled

LYSOPHOSPHATIDYLCHOLINE

LIPIDS

IN

EVERTED

in a concentration in phosphate

cholesterol

(0.15

in 6 or 8 experiments

of 0.8

buffer mM)

13H1

glycerol

[ 3H

1palmitate

LPC LPC

(6) (8)

sodium

Symbols recovery

(nmol/g

W)

mM

(LPC) SACS

and

containing

and

(N). 3Ii

substrate

JEJUNAL

micelles

taurocholate

in mucosal wet

INCORPORATION

OF

LABEL

(4.8

in either of oleic mM).

the glycerol acid Values

(0.6 are

or palmitate

mM), means

moiety.

monoacylglycerol k standard

I. lipids

weight/h)

TO

T

AND

37OC

was labeled

mixed

as in Table

tissue

AT

2879

f 202

793

6088

i- 470

2846

FFA +

88

” 250

PL

261

t 41

1759

1124

1803

’ 315

error

507

less dramatic than in the case of oleic acid (Fig. 3) and apparently did not involve an enhancement of esterification since most of the recovered label was in the free choles~rol form. Discussion Two recent studies have shown that phosphatidylcholine strongly inhibits intestinal micellar cholesterol and fatty acid absorption in vitro [1,2]. When care is taken to prevent hydrolysis of the compound it is also effective in vivo [ 11. Lysophosphatidylcholine, on the other hand, did not inhibit uptake in vivo in one study [l] suggesting that the intact phosphatidylcholine molecule is necessary for the inhibitory effect. The mechanism of the suppressor action of phosphatidylcholine may relate to its amphipathic properties and its ability to cause expansion of mixed bile acid-lipid micelles [6]. As mentioned earlier, an increase in micellar mass could reduce the coefficient of free diffusion of the micelles across the rate-limiting unstirred water layer, thus reducing the rate at which the lipids can gain access to the cell membrane. In support of this as a possible mechanism of action there is evidence that phosphatidylcholine cannot be absorbed intact 1191, except possibly for biliary phosphatidylcholine [ZO], and if this is true, its inhibitor action must relate to intraluminal events rather than intracellular events. The saturation phenomenon seen at the higher phosphatidylcholine concentrations (Fig. 1) needs further evaluation so that other unknown mechanisms may have to be considered. Lysophosphatidylcholine is not a swelling amphiphile [6], There is evidence in Table II and in other studies [21,22,23] that lysophosphatidylcholine is readily absorbed and, in the present study, its absorption was accompanied by a marked increase in the esterification of absorbed oleic acid (Fig. 3). In the presence of lysophosphatidylcholine the amount of absorbed oleic acid recovered in triacylglycerol greatly exceeded the amount recovered in free fatty acid and, in fact, the relative proportion present as free fatty acid actually decreased with increasing concentrations of the compound suggesting that the energetics of lysophosphatidylcholine action favored glycerol acylation rather than fatty acid uptake. Lysophosphatidylcholine also stimulated incorporation of oleic acid into the tissue phospholipids (PL, Fig. 3) but the effect was small, up to a maximum of 8% of the total lipid. The mechanism of action of the agent in this case was most likely due to its own direct acylation utilizing the absorbed fatty acid to form phosphatidylcholine as has been demonstrated in earlier studies [ 7,19,21, 221. Further evidence that lysophosphatidylcholine exerted an intracellular metabolic effect was obtained in the experiments in which the incubation temperature was lowered to near 0°C (Table I). In this case, in the absence of lysophosphatidylcholine the uptake of oleic acid was similar to that obtained at 37°C (Fig. 3), thus supporting earlier studies showing that fatty acid uptake occurs by a passive non-energy-requiring process [17,18]. However, the distribution of the label in the lipid fractions was quite different at 0°C most of it, as expected, appearing in the free fatty acid form. Adding lysophosphatidylcholine to the cold incubation fluid produced no effect in terms of enhancing

608

incorporation of the fatty acid into triacylglycerol or phospholipid, and it did not increase the total uptake as it did at 37’C. Thus, we conclude that lysophosphatidylcholine, in distinct contrast to phosphatidylcholine, has no direct effect on fatty acid uptake (a passive process) but has a marked effect on acylation of the fatty acid to glycerol. The increased glycerol acylation, in turn, could serve to enhance uptake by maintaining a concentration gradient for the free fatty acid between the incubation fluid and the cell cytoplasm. That lysophosphatidylcholine can be absorbed in vitro and hence exert the observed metabolic effects is clear from the results shown in Table II in which the compound itself was labeled in the glycerol or palmitate moiety. It should be noted that more than twice as much of the palmitate label was recoverd in the tissue lipids than the glycerol label even though, for unknown reasons, a considerable portion (approx. 19%) of the palmitate was unaccounted for in the fractions isolated. If the absorption rates of the two compounds were similar then some of the lysophosphatidylcholine was hydrolyzed with the loss of glycerol. The absorbed palmitate label was incorporated mostly into triacylglycerol (Table II). The amounts of label recovered in the phospholipids were similar regardless of whether the paimitate or glycerol was labeled and, in both cases, the amounts were several times greater than those found in the earlier experiments utilizing labeled oleic acid whether in the presence or absence of unlabeled lysophosphatidylcholine (Fig. 3). The results agree with those of Nilsson [ 211 who showed that labeled lysophosphatidyl~holine was more readily incorporated into lymph ehylomicrons than was labeled palmitie acid when the compounds were fed to “lymph fistula” rats. Figs. 1 and 2 are the results of identical experiments except that in the experiments of Fig. 2 the incubation mixture contained unlabeled cholesterol in addition to the other components listed. Comparing the left-most points in the two figures one can seen that cholesterol, in the one concentration used, suppressed oleic acid uptake by approximately 50% in the absence of phosphatidylcholine. Cholesterol is classified as a non-swelling amphiphile which solubilizes in the mixed bile acid-lipid micelle by interdigitating with the other micellar components without increasing the dimensions of the micelle [6]. Although there is disagreement on this point [ 241 cholesterol would add to the micellar weight and hence possibly reduce the coefficient of free diffusion of the micelle through the unstirred water layer as with phosphatidylcholine. A similar, though less pronounced, inhibitory effect of cholesterol on oleic acid uptake can be noted in the recent in vitro experiments of Rodgers and O’Connor [I] even though, in their experiments, the jejunal sacs were incubated for only five minutes. [ 3H]Cholesterol uptake in the present, study (Fig. 4) was measured only in double isotope experiments simultaneously with [ “C]oleic acid and only in the presence and absence of lysophosphatidylcholine (the phosphatidylcholine effects on cholesterol uptake were reported previously [2]). It was interesting that lysophosphatidylcholine bad o&y a minimal effect on cholesterol uptake and no effect on cholesterol esterification (Fig. 4). Although the increased uptake was significant at lysophosphatidylcholine concentrations of 0.8 and 1.2

mM it was not sustained at a higher concentration. It is possible, in fact, that the small increases observed were exaggerated or even spurious because of the spillover of the i4C label into the “H channel of the scintillation spectrometer in these double isotope experiments. An effort was made to correct for the spillover using the external standard method but a slight error could easily have accounted for the observed increases, particularly since the uptake of [‘“Cloleic acid increased at the same time (Fig. 3). Since cholesterol esterification was unchanged by the presence of lysophosphatidyl~holine and cholesterol uptake was only minimally affected, if at all, we have further support for the indea that the major influence of lysophosphatidylcholine in this study was to stimulate acylation of absorbed fatty acid to triacylglycerol with increased uptake of fatty acid probably occurring secondarily. In other words, its effects are best explained in terms other than assuming that it caused an alteration in micellar composition. Its effects then are intracellular rather than extracellular in contradistinction to phosphatidylcholine. The physiological significance of the divergent effects of the two phospholipids in vitro must be considered in the light of other studies showing that the predominant form of phospholipid recovered in,intestinal content in vivo is the lyso- form [ 81. Thus phosphatidylcholine may not suppress cholesterol and fatty acid absorption in vivo except possibly when it is given in amounts large enough to saturate the enzyme or in the form of an analogue which resists the action of the enzyme as has been shown recently to be the case for the diether analogue [ 11. Experiments are in progress to re-examine some of the possible mechanisms of action of phosphatidylcholine and lysophosphatidylcholine in terms specific to cellular uptake using short incubation times and avoiding the complications associated with the metabolic interconversions seen in the present study. Acknowledgements This research was supported by Grant No. 03189 from the National Institute of Arthritis, Metabolism and Digestive Diseases and by Grant No. 23.07 from the Medical Research Foundation of Oregon. The expert secretarial assistance of Alice Fitzgerald is gratefully acknowledged. References 1

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