absorption of oleic and palmitic emulsions micellar solutions

Absorption of Oleic and Palmitic Acids

from Emulsions and Micellar Solutions

W. J. SIMMONDS,T. G. REDGRAvE,and R. L. S. Waux

From the Department of Physiology, The University of Western Australia,Nedlands, Western Australia 6009

A B S T R A C T A lipid mixture (monoolein, oleicacid-1-14C, and palmitic acid-9,10-3H) was infusedintraduodenally at a steady rate for 8 hr in fasted,unanesthetized rats. The same dose of lipid wasgiven together with pure conjugated bile saltseither as an emulsion, 2.5 mmbile salts, or as amicellar solution, 10 mmbile salts. The emulsioncontained very little or no micellar lipid. Thoracicduct lymph was collected and in some experimentsbile and pancreatic juice were drained to the ex-terior. After 4-5 hr infusion the same steadylymphatic output of radioactive fatty acids wasobtained with emulsion as with micellar solution.It was concluded that absorption of fatty acidcould proceed efficiently in the virtual absence ofmicellar solubilization. In rats with biliary pluspancreatic fistulae, labeled triglyceride was ab-sorbed poorly relative to free fatty acids in thesame emulsified particles. This suggested thatfatty acids were transferred to the absorptive cellsin monomolecular solution and not as emulsionparticles.

Substitution of a synthetic nonionic detergentfor bile salts in lipid mixtures given to rats withbiliary and pancreatic fistulae did not affect thelymphatic output of radioactive fatty acids. Thisindicated that mucosal esterification of labeled freefatty acids was normal in the absence of bile salts.The physical state of the lipid did not affect thepathway of absorption. Finally, comparison of theincreased output of esterified fat in the lymph with

Address requests for reprints to Dr. W. J. Simmonds,Department of Physiology, The University of WesternAustralia, Nedlands, Western Australia 6009.

Received for publication 16 August 1967 and in revisedform 27 December 1967.

the output of labeled fat suggested that fat ab-sorption did not greatly affect the turnover ofendogenous, unlabeled fat. Results were consistentwith the view that most of the endogenous lymphfat comes from reabsorbed biliary lipid.

INTRODUCTIONDuring digestion and absorption, lipids in the lu-men of the intestine form a complex physicochemi-cal system. For example, the free fatty acids maybe present in unstable oil droplets, in emulsifiedparticles, perhaps in liquid crystalline aggregates,in mixed micelles, or in monomolecular solution.There is evidence that lipids are absorbed in sig-nificant amounts only when in a fine state of dis-persion as micelles or by diffusion as single mole-cules (1). It is possible that a limiting factor inabsorption might be the rate of transfer of mole-cules from the, very large aggregates found in oilglobules or even in finely emulsified particles tothe much smaller micellar aggregates or to mono-molecular dispersion (2). While the kinetics oftransfer of molecules between the different statesof aggregation can be more rigorously studied invitro, preliminary work in vivo seems desirable.Limiting factors in a closed in vitro system maywell be different from those in the open in vivosystem in which there is steady removal of ab-sorbed products (3).

This paper reports experiments which com-pared the absorption in lymph fistula rats of twofatty acids (palmitic and oleic acid) from a mix-ture of fatty acids and monoglycerides. The samemixture was presented either in micellar solutionwith bile salts or as a fine emulsion. In the latter

The Journal of Clinical Investigation Volume 47 1968 1015

case the bile salts were below the critical micellarconcentration (CMC) in the infusate. In the in-testine, mixture with endogenous bile salts mightstill allow formation of some micellar phase butthis was controlled by the use of rats with com-bined biliary and pancreatic fistulae.

The aims of the experiments were (a) to com-pare absorption rates for the same fatty acid mix-ture from micellar solution and from emulsionwith virtually no micellar phase; (b) to compareabsorption rates with two types of micellar solu-bilizer, bile salts and Pluronic F68, a nonionicdetergent; and (c) by giving increasing concen-trations of emulsion to see if there was any lag inabsorption attributable to delay in transfer of fattyacid from emulsified particles to micellar or mono-molecular dispersion. An important feature of theexperimental procedure was the steady infusionof lipid into the intestinal lumen for 8 hr so thatabsorption rates could be compared during thelatter half of the experiment when a fairly steadystate of absorption had been reached.

METHODSGeneral procedure. Male albino rats of Wistar strain,

interbred locally for 10 yr and weighing 200-220 g, wereused. Operations were done under ether anaesthesia af-ter fasting the animals overnight. The abdominal thoracicduct was cannulated according to the method of Boll-man, Cain, and Grindlay (4) with a polyethylene can-nula (I.D. 0.5 mm). In some rats the bile and pancreaticjuice were drained through an elastic silicone cannulatipped with polythene (I.D. 0.25 mm). The fine tip was in-serted between the junction of common bile and pan-creatic ducts and the duodenal wall. In all rats an elasticsilicone tube was passed through a small incision in thestomach into the duodenum and its tip was secured nearthe entrance of the bile duct.

After operation rats were transferred to restrainingcages. Thereafter NaCl solution, 0.9 g/100 ml, contain-ing KCl, 30 mg/100 ml, was infused continuously throughthe duodenal cannula until the end of the experiment, ex-cept when it was replaced by lipid mixtures. The volumeinfused was always 3 ml/hr. The first infusion of lipidwas given 48 hr after operation and, in many animals, asecond test was performed on the following day. Hourlylymph samples were collected into heparinized, graduatedcentrifuge tubes for the hour before lipid infusion andduring the 8 hr lipid infusion; then a 4 hr lymph col-lection followed by an 11-12 hr collection made up theovernight lymph recovery.

The infusate contained monoolein (MO), oleic acid(OA; Ci8:,) and palmitic acid (PA; Cie:o) in molar ra-tio 2: 2: 1, with some exceptions noted later. Stock solu-tions of lipid, kept at 5°C were mixed in the desired pro-

portions and tracer amounts of oleic acid-1-'4C and pal-mitic acid-9,10-3H were added to give OA a final specificactivity of about 12 X 10' dpm/umole, and PA about 30 X103 dpm/,umole. On the day of the experiment the re-quired volume of lipid mixture was taken and the solventwas completely evaporated under a stream of nitrogen.Pure bile salts or the nonionic detergent, Pluronic F68,in sodium phosphate buffer, 0.15 mole/liter in Na+, pH6.3 ± 0.1, was mixed with an equal volume of 0.9% NaClsolution and added to give the desired final concentra-tions of detergent and lipid. The phosphate buffer wasdiluted with saline to avoid diarrhea after prolongedadministration into the duodenum (5).

For micellar solutions with bile salts the final concen-tration of conjugated bile salts was 10 mmoles/liter andwith Pluronic F68 the concentration was 70 mg/ml(about 9 mmoles/liter). The final concentration of lipidmixture was about 7 mmoles/liter. The mixture wasstirred with a magnetic chuck under nitrogen at 37° Cuntil isotropic and kept at 37-42°C until used. The mi-cellar solutions remained transparent for long periods insealed vessels at 37°C and those made with MO, OA, andPA in molar ratio 2: 2: 1 were also stable at room tem-perature. Ultracentrifugation, 7 X 107 g-min at 37°C,showed that all the labeled fatty acid was in micellarsolution.

For emulsions with bile salts the final concentration ofbile salts was 2.5 mmoles/liter and with Pluronic F68the final concentration was 5 mg/ml (about 0.6 mmole/liter). For most experiments the total lipid concentrationwas 7 mmoles/liter. The material was insonated with aBranson Sonifier (40 w at 20,000 cycle/sec) for 5 min toproduce a fine emulsion. This was stable at 37°C forseveral days when made with MO, OA, and PA in molarratio 2: 2: 1, but was always prepared freshly on the dayof the experiment. Samples of infusate taken at the be-ginning, middle, and end of the infusion period wereanalysed for radioactivity to verify stability. The analysesagreed within 6% or less. A sample was taken at themiddle of the infusion period for estimation of totalsaponifiable fatty acid.

Ultracentrifugation, 7 X 107 g-min at 370C, of infusatesemulsified with 2.5 mmbile salts, showed that the con-centration of labeled fatty acid in the isotropic phasewas approximately 4 X 10' mole/liter for oleic acid and2 X 10' mole/liter for palmitic acid. This would be con-sistent with monomolecular solution, i.e., absence of amicellar phase.

As a further test, the following experiment was per-formed: The standard lipid mixture MO-OA-PA, molarratio 2: 2: 1, was emulsified by insonation in buffer solu-tion alone, and in buffer plus three different concentrationsof bile salts (1.0, 2.5, and 7.5 mmoles/liter). The emul:sion in the absence of bile salts was unstable. The iso-tropic phase was separated from each emulsion by cen-trifugation, and to 10 ml of isotropic phase was added 3mg azobenzene. The mixture was insonated, 40 w at20,000 cycle/sec, and the optical density measured witha Beckman DB spectrophotometer at 320 mu againstphosphate buffer. The optical densities were 0.025, 0.025,

1016 W. J. Simmonds, T. G. Redgrave, and R. L. S. Willix

0.025, and 0.70 for isotropic phases with 0, 1.0, 2.5, and7.5 mmbile salt mixture. The optical density for 10 mMbile salt solution alone in phosphate buffer against a phos-phate buffer blank was 0.05. Thus the isotropic phase of alipid mixture in 2.5 mmbile salts solubilized no moreazobenzene than did the isotropic phase from a mixturewith 1 mmbile salt or with none. The amount of azo-benzene solubilized was very small compared with theamount solubilized by the isotropic phase from a lipidmixture in 7.5 mmbile salts in which about 6.5 itmole oftotal lipid/ml was micellar. It seemed, therefore, that ifthere were any micellar phase in the emulsified infusatewith bile salt concentration 2.5 mmoles/liter, the amountmust be very small. A similar conclusion was reached inexperiments on micellar solubilization of radioactive fattyacids, see Results. Although such experiments showedthat the CMCfor the lipid mixture was close to 2.5 mMbile salts, so that a lower bile salt concentration mighthave been preferable for emulsified infusates, it was foundthat emulsions made with lower bile salt concentrationssometimes became unstable, particularly if lipid mixtureswere rich in PA. It seemed preferable to accept the pos-sibility of a trace of micellar phase in order to avoidgross errors which would follow breaking of the emul-sion, particularly if this occurred in the infusion tubing.

Materials. Glycerol-1-monooleate (Monoolein) 1 wasshown by thin-layer chromatography (TLC) to containa small amount of diglyceride and free fatty acid. By gas-liquid chromatography (GLC) the percentage fatty acidcomposition was-C12:0, 1; Cie:,, 5; Cis:i, 85; C18:2, 5.Oleic acid 2 was 98.5% pure and palmitic acid 3 was 99%pure by GLC. Radiochemical purity of oleic acid-1-"C,4was certified by the suppliers as 95-98% pure C18: 1 ofwhich 5-7% was elaidic acid. On TLC in a mixture withunlabeled MO, OA, and PA 98%o of the radioactivity wasrecovered in the free fatty acid band. Palmitic acid-9,10-3H,4 was certified by the suppliers as 96% pure. On TLCin a mixture with unlabeled MO, OA, and PA 95%o ofthe radioactivity was recovered in the free fatty acidband. Glyceryl tri (oleate-1-"C) ,4 was certified by thesuppliers as 98-99% pure. The labeled and unlabeledlipids were all used without further purification.

The bile salt mixture consisted of sodium taurocholate(NaTC) and sodium taurodeoxycholate (NaTDC) inmolar ratio 4:1. The NaTC2 moved as a single spoton TLC and was used as supplied. The NaTDCwas syn-thesized from taurine 3 and deoxycholic acid 5 by themethod of Norman, modified by Hofmann (6). The de-oxycholate moved as a single spot on TLC. The syn-thetic NaTDC showed only a trace of free bile salts.The nonionic detergent, Pluronic F68,6 has been de-scribed as a block copolymer of propylene and ethylene

1 Calbiochem, Los Angeles, Calif.2 Koch Light Laboratories, Colnbrook, Bucks, England.3 Eastman Organic Chemicals, Rochester, N. Y.4 Radiochemical Centre, Amersham, England.5 Nutritional Biochemical Corporation, Cleveland, Ohio.6 Kindly donated by Wyandotte Chemicals Corp., Wyan-

dotte, Mich.

oxides of average molecular weight 8000 (7) and wasused as supplied.

Analytical. TLC was carried out on 0.25 mmlayers ofSilica Gel G 7 on plates reactivated at 110°C for 1 hr be-fore use. Up to 250 ,g of material was applied and de-veloped for 15 cm. For glycerides the solvent system washexane-ether-acetic acid, 80: 20: 2 v/v/v. For bile saltsethylacetate-methanol-acetic acid, 70: 20: 10 v/v/v, wasused. GLC of fatty acids was carried out in 4-ft glasscolumns of 20% diethylene glycol succinate on Gas-ChromCLA8 support at 190-200'C, with argon as carrier gas anda strontium 90 ionization detector. Analyses of standardmixtures (NIH standard mixture) agreed with thespecifications with a relative error of less than 6% formajor components and less than 20%o for minor compo-nents (less than 10% of total).

Radioactivity was determined on aliquots of alcohol-ether extracts of lymph and infusate which had been pre-pared for analysis of esterified fat (8). The solvent wasevaporated and radioactivity was measured in a solutionof 2,5-diphenyloxazole, 4 g/liter, and 1,4-bis[2-(5-phenyl-oxazolyl)]benzene, 0.05 g/liter, in toluene, using a Nu-clear-Chicago counter. Quenching correction was madeby the channels ratio method (9) but the degree ofquenching was usually less than 10%, so that correctionswere small. Samples of infusate were counted at thesame time as samples of lymph and recoveries of radio-activity in lymph were expressed as a percentage of 14Cand 3H infused. The total radioactivity infused was alwaysabout 0.3 uc of '4C (OA) and about 0.4 ,uc of 'H (PA).

Total esterified fatty acid in lymph and infusate weremeasured by the method of Stern and Shapiro (8). Totalfatty acid in the infusate was measured, after saponifica-tion, by the method of Frazer (10).

Micellar solubilization. Lipid mixtures of the requiredcomposition were made up in buffered surfactant solu-tion as described above for infusates. All mixtures wereinsonated for 5 min (Branson Sonifier, 40 w, 20,000cycles/sec at 37°C. 10 ml was transferred immediately toan ultracentrifuge (Spinco L-2 preparative) without cool-ing and spun for 7 X 107 g-min at 37°C. 5 ml of the lowerisotropic phase was withdrawn without remixing, in-jected into 15 ml of ethanol-diethyl ether-n-hexane, 1:1: 1v/v/v, and the lipid extracted by the method of Blanken-horn and Ahrens (11). The solvent was evaporated andradioactivity measured as described above. A 5-ml sampleof the original mixture before centrifugation was similarlytreated. Control runs showed that less than 2%o of radio-activity was lost in preparation of the emulsion for cen-trifugation. The volume of supernatant oil and emulsionafter centrifugation was negligible and there was nosediment. Fatty acid mass was estimated from radio-activity and the known specific activity.

RESULTS

Micellar and nonmicellar absorption. Micellarand nonmicellar absorption were compared by in-

7 E. Merck, Darmstadt, Germany.8 Applied Science Laboratories Inc., State College, Pa.

Absorption of Emulsified and Micellar Fatty Acids 1017

fusing the same lipid mixture (MO-GA-PA inmolar ratio 2: 2: 1) in about the same concen-tration, 7 mmoles/liter, varying only the concen-tration of bile salts or nonionic detergent. The re-sults are summarized in Fig. 1. It can be seen thatin all experimental groups a steady infusion oflipid mixture intraduodenally produced within4-5 hr a steady rate of transfer of the two labeledfatty acids into lymph.

In rats with intact bile ducts, absorption was thesame whether fatty acids were infused in mono-olein-bile salt micelles, 10 mmbile salts, or inmonoolein-bile salt emulsion, 2.5 mmbile salts(compare Fig. 1 A, upper and lower). Micellarsolubilization experiments, which are presented

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later, showed that 2.5 mmoles/liter was close to orbelow the critical micellar concentration for thebile salt mixture. There was direct evidence thatthe infusate emulsified with bile salts contained nosignificant micellar phase, as set out under Meth-ods. Ultracentrifugation of the emulsified infusateshowed that the concentration of labeled fatty acidin the isotropic phase was 3.5 x 10-5 mole/literfor oleic acid and 1.7 x 105 mole/liter for pal-mitic acid, which would be consistent with mono-molecular solution.

Although there was virtually no micellar fattyacid in the infused bile salt emulsion, experimentsin rats with intact bile ducts did not exclude pos-sible micellar solubilization in the intestinal lumen

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FIGURE 1 Absorption of micellar and emulsified lipid. In all experiments a mixture of monoolein-oleic acid-14C-palmitic acid-3H in molar ratio 2: 2 :1, concentration 7 mmoles/liter, was infused intothe duodenum at approximately 21 ,umoles/hr. In the upper graphs the lipid was in micellar solutionin a mixture of pure sodium taurocholate-sodium taurodeoxycholate, molar ratio 4: 1, 10 mmoles/liter(A and B), or in nonionic detergent, Pluronic F68, 70 mg/mI (C). In the lower graphs the lipid wasemulsified in the same bile salt mixture, 2.5 mmoles/liter (A and B) or in Pluronic F68, 5 mg/ml (C).The vehicle was phosphate buffer-saline, 0.15 mole/liter in Nat, pH 6.3 - 0.1. In A, upper and lower,the rats had intact bile ducts; in B and C, upper and lower, bile and pancreatic juice were drained tothe exterior.

Hourly output of radioactivity in lymph is expressed as per cent of oleic acid-"4C, *-*, or palmiticacid-3H, *-*, infused per hour. The ratio of percentage absorption, oleic acid-palmitic acid, isshown above, 0-0. Each graph is the average of two experiments except C, (lower) which is fora single experiment.


by endogenous bile salts. This was unlikely in ratswith cannulae draining both bile and pancreaticjuice (Fig. 1 B). Absorption of fatty acids,whether micellar or emulsified, was somewhat lessefficient than in rats with intact bile ducts. Thispossibly reflects the difficulty of maintaining thedoubly depleted animals in optimal condition.However, there was still no difference betweenabsorption of micellar and emulsified fatty acids(compare Fig. 1 B, upper and lower). It shouldalso be noted that fatty acids were absorbed frommicellar solution in a nonionic detergent, PluronicF68, as efficiently as from bile salt micelles (com-pare Fig. 1 B and 1 C, upper). The number ofexperiments was small but the results were quiteconsistent (Table I).

It should perhaps be emphasized that the emul-sion made with Pluronic F68 (Fig. 1 C, lowergraph) contained a substantial micellar phase.Therefore, comparison of upper and lower graphsin Fig. 1 C gives no information on micellar vs.nonmicellar absorption in the presence of PluronicF68. Emulsions were unstable when made with lowconcentrations of Pluronic F68, at or below theCMC for detergent plus the usual mixture ofmonoolein, oleic acid, and palmitic acid. A detailedcomparison with Pluronic F68 was, therefore, notattempted.

In rats with combined bile and pancreatic fistu-

TABLE IHourly Lymphatic Output of Radioactive Fatty Acid*

Lymphatic output

Treatmentl Infused Hr. .5 6 7 8

Am 11.9±0.2 7.2i0.4 7.3±40.5 7.1±0.5 7.1±0.8Ae 12.3--0.5 7.6±+0.5 8.040.7 8.14±0.4 8.2 ±0.4Bm 11.9 40.04 5.4 ±0.5 5.5 i0.3 5.7 ±0.1 6.3 ±0.2Be 11.9+0.3 5.7±0.3 6.640.4 6.4±0.1 6.6+02.cm 12.6±1.4 6.341.1 7.1+1.5 6.2±0.3 6.4±0.8

* Calculated, as Mmoles/hr, from hourly output of 14C (cpm) X speci-fic activity cpm/gmole of unesterified oleic acid-4C infused plus hourlyoutput of 'H X specific activity of unesterified palmitic acid-3H infused.* Am, four experiments, intact bile ducts. Micellar infusate in 10 mmbile salts; monoolein-oleic acid-palmitic acid molar ratio 2:2: 1 in twoexperiments and 2:1:2 in the other two. Results as mean ± SEM.Ae. four experiments, intact bile ducts. Emulsion in 2.5 mmbile salts,two experiments with monoolein-oleic acid-palmitic acid molar ratio2:2:1 and 2:1:2 in the other two. Results as mean ± SEM. Bm, twoexperiments, biliary plus pancreatic fistula. Micellar infusate in 10mmbile salts; monoolein-oleic acid-palmitic acid molar ratio 2:2:1.Results as mean ±- half range. Be, two experiments same as for Bmexcept that emulsion in 2.5 mMbile salts was infused. Cm, two experi-ments, same as for Bmexcept that Pluronic F68, 70 mg/ml, was used asmicellar solubilizer. Total radioactive free fatty acid infused, inpumoles per hour.

TABLE I IAbsorption of Emulsified Triolein in Rats with

Pancreatico Biliary Fistulae*

Triolein-14C Oleic acid-14C

Rat No. 14C 3H "4C 3H

1 5.2 32.4 55.4 47.32 24.1 42.5 63.9 53.53 17.7 30.9 41.6 35.54 9.6 30.5

* On the left hand side are shown results when the mixtureinfused was monoolein, glycerol tri(oleate 1 14C), oleic acid(unlabeled), and palmitic acid 9,10-3H in molar ratio forfatty acid chains of 2:3:1:1, as a fine emulsion in 2.5 mMbile salts (rat 4) or Pluronic F68, 5 mg/ml (rats 1, 2, and 3).On the right hand side the mixture infused in rats 1 and 3was monoolein, oleic acid 1-14C, and palmitic acid-9,10-3Hin molar ratio 2:2:1. In rat 2 the mixture given on the 2ndday, right-hand side, was the same as on the 1st day ex-cept that the free oleic acid was labeled and the triolein wasnot. Absorption is calculated as per cent of radioactivityinfused per hour, averaged for hr 6, 7, and 8 of infusion.The total lipid infused was about 21 jumoles/hr containingabout 30 smoles saponifiable fatty acid in the emulsionscontaining triolein and 21 /Amoles saponifiable fatty acidwhen no triolein was present.

lae when triolein esterified with oleic acid-1-14Creplaced half the oleic acid in the lipid mixture,the remainder being unlabeled, the absorption of14C label was greatly decreased but the absorptionof palmitic acid-3H from the same emulsified par-ticles was only slightly decreased (Table II). Theoleic acid-14C which appeared in the lymph wasprobably absorbed after hydrolysis of triolein-"4Cby residual lipase. Although rats 1 and 3, TableII, received the same total lipid, in jumoles, on bothdays, the molar ratio of free to esterified oleicacid and the total saponifiable fatty acid differed inthe two infusates. However, similar results wereseen in rat 2, in which the infusates differed onlyin the labeling with 14C of triglyceride or freeoleic acid, respectively. These experiments sug-gested that the efficient absorption of emulsifiedfree fatty acid with virtually no micellar phase wasdue to absorption of fatty acid as a monomolecularsolution rather than as emulsified particles.

Varying concentration and composition of lipidmixture. The results presented so far were con-sistent with absorption of fatty acid by diffusionfrom monomolecular solution, whether the bulkof the fatty acid was present as micellar aggregates


or as emulsified droplets. Palmitic acid was trans-ferred to the lymph somewhat more slowly thanoleic acid, but here were no differences betweenmicellar and emulsified infusates in the absoluteor relative absorption rates of the two fatty acids.

This suggested that transfer of fatty acid fromemulsified droplets to molecular dispersion wasnot a rate limiting step at low concentrations. Theeffect of increasing the concentration of emulsifiedlipid was measured in a small series of experi-ments. Two lipid mixtures were tested with mono-olein, oleic acid, and palmitic acid in molar ra-tios 2: 2: 1 and 2: 1: 2, respectively. Since earlierexperiments (see Fig. 1) suggested that the ef-fects of combined biliary and pancreatic fistulaemight decrease the capacity of lymph fistula ratsto handle an absorptive load the biliary and pan-creatic ducts were left intact. Some micellar lipidphase might, therefore, have formed in the intes-tine, although none was present in the infusate.Table III shows the absorption into lymph of la-beled fatty acid during the last 3 hr of infusion,when a steady state had been reached. The per-centage of infused radioactivity which was ab-sorbed did not decrease with increasing absorp-tive load. This indicated that the mass of fattyacid absorbed per hour increased pari passu withthe load infused. For each lipid mixture absorp-

TABLE I I ISteady-State Output of Labeled Fatty Acids in Lymph*

Output as per cent infused

High oleic High palmitic

Total lipid Oleic Palmitic Ratio, Oleic Palmitic Ratio,concentration acid-14C acid-3H 14C/3H acid-14C acid-3H 14C/3H

mmoles/ml7 (M) 67 56 1.20 81 58 1.41

71 57 1.24 62 37 1.687 (E)§ 62 53 1.17 77 59 1.31

68 56 1.22 88 69 1.2514 (E) 85 77 1.(9 79 63 1.2728 (E) 87 73 1.20 75 55 1.38

* Hourly output ot oleic acid-1-14C and palmitic acid-9-, 10-3H as per centot radioactivity infused per hour averaged for hr 6, 7, and 8 of infusion.Results of 12 experiments on nine rats are shown. In all experiments onthe left-hand side (high oleic) the lipids were monoolein-oleic acid-palmitic acid in molar ratio 2:2: 1. Onthe right-hand side (high palmi-tic) are results for the ratio 2:1:2, i.e., the proportions of oleic andpalmitic acid were reversed. In the lower four experiments on eachside the lipids ere emulsified in 2.5 mMbile salts, sodium taurocholate-sodium taurodeoxycholate, 4:1). In the top two experiments on eachside the lipids were micellar in 10 mmbile salts.t M, micellar.§ E, emulsion.

tion of oleic acid relative to palmitic acid over afourfold range of concentration in emulsion wasthe same as in a micellar solution.

Endogenous lymph lipid. Infusion of a lipidmixture into the intestine increased the output ofesterified fatty acid in the lymph, a steady levelbeing reached within about 5 hr. Most of the fattyacid in lymph is esterified. An increase in esteri-fied fatty acid output represented the total increasein output of exogenous plus endogenous fatty acidwhereas recovery of infused radioactive fatty acidrepresented only the exogenous contribution.

In Fig. 2 A it can be seen that the esterifiedfatty acid output in lymph increased much morethan would have been expected from the recoveryof radioactive oleic acid, when micellar solutionsrich in bile salts were given to rats with intactbile ducts. That is, there was a substantial increasefrom endogenous sources. The same high concen-tration of bile salts had a smaller effect in ratswith biliary fistulae, Fig. 2 D, than in those withintact bile ducts, Fig. 2 A. The same concentrationof administered lipid when emulsified in a lowconcentration of bile salts had little effect whetherthere was a biliary fistula, Fig. 9 E, or not, Fig.2B.

The endogenous esterified fatty acid in lymphwas calculated for these experiments, as set out inTable IV. The effect of bile salts in rats with in-tact bile ducts was statistically significant (P <0.05 t test, 6 df, on means A and B). With suchsmall numbers a detailed treatment of the otherresults is not warranted but they raise some pointsof interest which are briefly considered in Dis-cussion.

Micellar solubilization. Emulsions were madewith lipids mixed in the same proportions as in theabsorption experiments, viz. MO-OA-PA in mo-lar ratios 2: 2: 1 and 2: 1: 2, but with varyingconcentrations of each of the two detergents, bilesalts and Pluronic F68. The fatty acid composi-tion of the micellar phase separated by high speedcentrifugation, 7 x 107 g-min at 370C, is shownin Fig. 3.

In these experiments a constant amount of lipidwas used to prepare the emulsion and the concen-tration of detergent was progressively increaseduntil most of the lipid was solubilized. The ob-jectives were strictly limited. Firstly, we wishedto determine approximately the CMCunder the


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FIGURE 2 Endogenous fat in lyImiph. Hourly output in lymph of total esterifiedfatty acid minus the fasting output in the hour before fat infusion, 0-0, andoutput of oleic acid-'4C, *-*, are expressed as per cent of amounts, respec-

tively, of saponifiable fatty acid and oleic acid-'4C infused intraduodenally per

hour. In all experiments the lipid mixture infused was monoolein-oleic acid-'4C-palmitic acid-3H, molar ratio 2: 2: 1 in half of each group A to F and 2: 1: 2 inthe remainder of each group, in sodium taurocholate-sodium taurodeoxycholatemixture, 10 mmoles/liter (micellar) or 2.5 mmoles/liter (emulsion), in phos-phate-saline, 0.15 moles/liter in Na+, pH 6.3 + 0.1. A, bile ducts intact, micellarlipid 21 Mtmoles/hr (4) (numbers in parentheses equal number of experiments);B, bile ducts intact, emulsion, 21 itmoles/hr (4); C, bile ducts intact, emulsion,42 Flmoles/hr (2); D, bile and pancreatic fistula, micellar lipid, 21 )Umoles/hr(2) E, bile and pancreatic fistula, emulsion, 21 /umoles/hr (2) ; and F, bileducts intact, emulsion 84 umoles/hr (2).

conditions used for preparing emulsions. The sol-ubilization curves indicated a CMIC a little above2.5 mmoles/liter for the bile salt mixture (N\aTC-NaTDC4:1) and about 0.1 mmnole/liter for thenonionic detergent, Pluronic F68. Fig. 3 does notshow the very low concentrations of radioactivefree fattv acid in the isotropic phase with bile salt,2.5 mmoles /liter and below, nor could these con-

centrations be estimated very accurately. In threemixtures with 1.0, 1.5. and 2.0 mar bile salts theconcentrations in the isotropic phase of oleic acid-w4C ere approximately 4 x 10-6, 1 x 10-5, and

5 X 10-6 mole/liter, respectively. In 2.5 mmbilesalts the concentration of oleic acid-14C was 4 X

10-5 mole/liter and that of palmitic acid was 2 x10-5 mole/liter. More detailed work would beneeded to establish the significance of this differ-ence from free fatty acid concentrations with lowerconcentrations of bile salts but the values at 2.5mar bile salts were still within the likely range formonomolecular solution of free fatty acid. Forreasons mentioned under Methods 2.5 mmi bilesalts was used for lipid emulsions rather thanconcentrations well below the CMC.

Absorption of Emulsified and Micellar Fatty Acids

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TABLE IVHourly Output of Endogenous Lipid in Lymph

Increase during infusion§

Groups Preinfusion$ Hr. .5 6 7 8 Total

A 17.5 ± 0.6 8.3 2.2 8.9 ± 3.3 6.6 + 1.8 7.3 ± 2.3 31.1 ± 8.9B 18.8 i 1.7 -0.3 i 1.4 0.1 i 1.6 0.8 i 0.4 2.4 ± 2.2 3.2 i: 4.8C 20.4 i 1.2 4.9 4 1.4 1.5 at 0.6 4.0 i 0.8 6.4 4± 0.8 16.7 i 3.5D 7.5 i 2.0 2.6 i 3.0 5.0 ± 0.4 2.4 i 0.5 2.9 4 2.0 12.8 i 4.2E 4.0 ±:2.4 -1.7±0.1 0.9±2.0 -0.3±1.7 1.5±0.6 21±4.7F 14.9 i 1.4 5.1 i 1.3 5.8 ± 9.3 1.5 ± 2.7 2.2 ± 0.9 14.0 ± 6.8

* Groups as in Fig. 2.Fasting output of esterified fatty acid in ,umoles per hour for 1-2 hr before intraduodenal infusion of lipid.

§ The preinfusion output of esterified fatty acid (endogenous) was subtracted from the total esterified fatty acid outputduring the specified hour of infusion, to give the increase in esterified fatty acid atributable to infusion. The portion ofthe increase due to exogenous fatty acid was calculated from recovery of palmitic acid-H3 X specific activity of infushedpalmitic acid plus recovery of oleic acid-'4C X specific activity of infused oleic acid, including monoolein. This assumedthe same efficiency of absorption of free, labeled oleic acid and unlabeled monoolein. The endogenous contribution wasobtained by subtracting the calculated increase of exogenous origin from the total increase in esterified fatty acid attribut-able to infusion. Mean ± SEMfor A and B (four experiments in each); mean + half range for C through F (two experi-ments in each).

Secondly, we wished to see if the molar ratio while the proportion of micellar to emulsified lipidof the two labeled fatty acids remained the same varied widely. This proved to be the case.in the micellar phase as in the total lipid mixture Finally, the nonionic detergent was a more effi-

A B

80-

60- me.mm.P * m..@4- -i.mm

40 _ * _ _.

6-

4-

2-

2 4 6 8 10 0.4 0.8 1.2 1.6BILE SALTS (j.moles/ml) PLURONIC F68 (CjmoleS/mI)

FIGURE 3 Micellar solubilization, in vitro. Total fatty acid concentration, mmoles/liter, in micellar solution calculated from the specific activity of oleic acid-"4C and pal-mitic acid-3H and the radioactivity in the isotropic infranatant fluid after centrifuga-tion, 7 X 107 g-min at 37°C. The total concentration of lipid was always 10 mmoles/liter, of which free fatty acids constituted 6 mmoles/liter in phosphate-saline, 0.15mole/liter in Na+, pH 6.3 ± 0.1. *, monoolein-oleic acid-14C- palmitic acid-8H, molarratio 2: 2: 1 and *, in molar ratio 2: 1: 2, respectively. At the top it can be seen thatoleic acid always constituted the same percentage of total labeled fatty acids in the mi-cellar phase, * and *, as in the starting material, dashed line, which formed the finalmixture of micellar and emulsion phases.


cient solubilizer on a molar basis for monoolein-fatty acid mixture than were the mixed bile salts.This seeming advantage might only hold for alimited range of conditions; for example, it will benoted that the shapes of the solubilization curvesare different (Fig. 3A and B). A more detailedstudy of the mixed Pluronic F68 micelle shouldprove interesting.

DISCUSSION

If a micellar phase was present in the bile saltemulsion the amount must have been extremelysmall, yet fatty acids were absorbed as effectivelyas from micellar solution. It is important to ex-clude the possibility that a significant micellarphase was formed from the emulsion in the intes-tinal lumen. In rats with intact bile ducts thismight have occurred by addition of endogenousbile salts or other amphipaths to the emulsion;but results were the same in rats with combinedbiliary and pancreatic fistulae. Even in such ani-mals it could be argued that absorption of fluidfrom the lumen could have raised the concentra-tion of administered bile salts above the CMC.However, no micellar fatty acid was found by cen-trifugation of undiluted intestinal contents takenafter infusion of bile salt emulsion for 3.5 hr.The rat is an unsatisfactory animal for detailedphysicochemical study of intestinal contents butit seems reasonable to conclude that if a micellarphase was present either in the lumen or in the bilesalt emulsions which were administered the amountof micellar lipid must have been very small.

Absorption of nonmicellar lipid from emulsionsmight proceed by uptake of particles or by diffu-sion from monomolecular solution. The latterseems much more likely. As Table II shows, pal-mitic acid-3H was absorbed much more rapidlythan triolein-14C from the same emulsified par-ticles, whereas emulsified free oleic acid-'4C wasabsorbed somewhat more rapidly than palmiticacid-3H. The 14C label which did appear in thelymph from triolein-14C emulsions could be ex-plained by residual lipolytic activity in the lumenliberating labeled oleic acid (12).

Absorption from a micellar solution was nofaster than nonmicellar absorption for the samelow dose of fatty acid, but it is possible that non-micellar absorption might be rate limited at a

higher dosage. For example, micellar aggregates

might serve as a reservoir of fatty acid to preventdesaturation of monomolecular solution when fattyacid molecules were being taken up rapidly intothe epithelial cell membrane from the adjacentlayer, a few molecules thick, of unstirred luminalfluid (13, 14). Further work on this possibility isneeded. In a small number of experiments no evi-dence of rate limitation was found when an in-creasing dosage of emulsion free of micellar phasewas administered to rats with intact bile ducts(Table II).

Morgan (15) found that in bile fistula rats,oleic acid finely emulsified with Pluronic F68 wasabsorbed at a rate comparable with the normaldietary load. In his experiments the concentrationof Pluronic F68, 0.6 mg/ml, was close to the CMCdetermined for a monoolein, fatty acid mixture inthe present experiments. However, lecithin wasalso present in Morgan's emulsions so that somelysolecithin might have been formed in the lumenas an additional micellar solubilizer.

Calculations suggest that diffusion could ac-count for transfer of the normal dietary load offat, as fatty acid, from lumen to intestinal epi-thelium (14, 16). Although absorption of dietarytriglyceride is known to be defective when micel-lar solubilization is defective, the concurrent de-fects in emulsification and lipolysis have not beenruled out as rate limiting factors; equilibration be-tween large globules and single lipid molecules insolution might be very slow. The emulsion pre-pared by insonation in the present experimentsmay have been finer than that attained normallyduring digestion of lipid. The influence of the in-terfacial area of emulsion on exchange rates withmonomolecular solution has yet to be determined.The present experiments, however, suggest quitestrongly that a minimal or absent micellar phaseneed not interfere with transfer of lipid from in-testinal lumen to lymph.

Micellar solubilization of itself may confer nogreat advantage in absorption of lipid moleculessuch as ionized fatty acids which have a definitethough low solubility as single molecules in aque-ous solution. Yet the same may not be true forvirtually insoluble lipid such as cholesterol. Cho-lesterol absorption is negligible in the absence ofbile salts (17) and it may be that uptake aftercollision of micelles with the cell membrane is theonly truly significant mode of transfer of highly


insoluble lipid from lumen to cell. Experimentssimilar to those described for fatty acid will nextbe undertaken with cholesterol. While the natureof the micellar solubilizer seems unimportant forfatty acid absorption (Fig. 1) it will be of interestto see if this is also true for cholesterol absorption.

The results with micellar solution in nonionicdetergent support other evidence (15) that bilesalts are not necessary for efficient reesterificationduring absorption. Moreover, since the lipid wasmicellar the suggestion of Saunders and Dawson(18) that fine emulsion promotes pinocytosis whichin turn promotes lymphatic transport is not ap-plicable to these experiments.

Whether micellar or nonmicellar, oleic acid wasabsorbed somewhat more rapidly relative to theinfused load than was palmitic acid. Evidence fromthe present experiments does not suggest that thedifference can be attributed to differences in mi-cellar solubility or in partition between micellarphase and emulsion particles. There is very littledata on solubility of fatty acids in monomoleculardispersion under the conditions found in the in-testinal lumen and an attempt is being made at invitro measurements. In vivo, the steady-state in-fusion method offers an opportunity to discrimi-nate between intraluminal and intracellular factorsdetermining differences in absorption of differentlipids. It will be necessary to use animals largerthan rats to allow serial sampling of intestinal con-tents for physicochemical measurements.

Endogenous fatty acid in the lymph. Nearlyall the fatty acid in lymph is esterified, whether ofdietary or nondietary (endogenous) origin. Evi-dence is accumulating that a major part of theendogenous fatty acid in fasting rats is absorbedinto the lymph from biliary phospholipids in theintestinal lumen (19, 20). After fat feeding thereis a substantial dilution of labeled dietary fat byunlabeled endogenous fat (21, 22). However, inconcluding that absorption of dietary fat promotesthe turnover of endogenous fatty acid the basal,fasting contribution needs to be taken into account.In groups of thoracic duct fistula rats studied inthis department during the past 10 yr, the averagefasting output of ester fat has always been closeto 20 ,Eq/hr, i.e., about 120-150 mg of endoge-nous fatty acid per day. This is similar to the totalamount of unlabeled fatty acid found by Karmen,Whyte, and Goodman (21) in 24-hr collections of

lymph after fatty meals. They measured basal out-put but the value, 5-20 mg/24 hr, was much lowerthan usually reported.

In the present work the amount of unlabeledfatty acid in lymph increased less with increasingabsorptive load of labeled fatty acid (Figs. 2 B, C,and F) than with a small dose of lipid solubilizedin a high concentration of bile salts (Fig. 2 A).The latter effect was probably due to increasedbiliary secretion of phospholipid into the intestinallumen, since the same lipid-bile salt mixture hada smaller effect on endogenous fat in rats withbile fistulae (Fig. 2 D).

The present experiments were not primarilydesigned to see whether absorption of dietary fatincreased the endogenous output in lymph. Itwould be premature to stress the apparent lack ofeffect in these few experiments compared with theincreased endogenous contribution inferred byother workers (21, 22). The weaknesses of thepresent experiments were the use of unlabeledmonoolein in the lipid mixture and the lack of dataon the distribution of fatty acid by radioactivityand by mass (gas-liquid chromatography) in thelymph (21). However, there are two useful fea-tures of the present approach. The analysis ofserial lymph samples allows the basal, fastingendogenous contribution to be compared with anychanges during fat feeding and shows the timingof any increase in the endogenous component.The steady infusion of test lipid mixture allowsthe dilution of exogenous lipid to be measured un-der conditions of steady transfer of absorbed fatfrom intestinal epithelium into lymph. Steady-state experiments with full lipid analysis in nor-mal and bile fistula rats should help to settle theissue.

ACKNOWLEDGMENTSWewish to thank Miss Morag Long (English SpeakingUnion Vacation Scholar) and Mr. W. J. Strickland(National Heart Foundation of Australia VacationScholar) who participated in aspects of the work andMiss V. Orton who gave valuable technical assistancethroughout.

The nonionic detergent, Pluronic F68, was a gift fromWyandotte Chemical Co., Wyandotte, Mich.

This work was supported by grants from the AustralianResearch Grants Committee, The National Health andMedical Research Council of Australia, and the MedicalResearch Grants Committee of the University of WesternAustralia. Dr. Redgrave is a Life Insurance Medical Re-


search Fund of Australia and New Zealand fellow. Dr.Willix is an Australian Research Grants Committee post-doctoral fellow.

REFERENCES

1. Senior, J. R. 1964. Intestinal absorption of fats. J.Lipid Res. 5: 495.

2. Hofmann, A. F. 1966. A physicochemical approach tothe intraluminal phase of fat absorption. Gastroenter-ology. 50: 56.

3. Feldman, E. B., and B. Borgstrom. 1966. Absorptionof sterols by intestinal slices in vitro. Biochim. Bio-phys. Acta. 125: 148.

4. Bollman, J. L., J. C. Cain, and J. H. Grindlay. 1948.Techniques for the collection of lymph from theliver, small intestine or thoracic duct of the rat.J. Lab. Clin. Med. 33: 1349.

5. Redgrave, T. G. 1967. The absorption of micellarlipid into the lymph of unanesthetized rats. Quart. J.Exptl. Physiol. 52: 130.

6. Hofmann, A. F. 1963. The preparation of cheno-deoxycholic acid and its glycine and taurine conju-gates. Acta Chem. Scand. 17: 173.

7. Schmolka, I. R., and A. J. Raymond. 1965. Micelleformation of polyoxyethylene-polyoxypropylene sur-factants. J. Am. Oil Chemists' Soc. 42: 1088.

8. Stem, I., and B. Shapiro. 1953, Rapid and simplemethod for determination of esterified fatty acids andfor total fatty acids in blood. J. Clin. Path. 6: 158.

9. Hendler, R. W. 1964. Procedure for simultaneousassay of two 8-emitting isotopes with the liquid scin-tillation counting technique. Anal. Biochem. 7: 110.

10. Frazer, A. C. 1960. Daily fatty acid excretion(Broadsheet No. 28). Association of Clinical Pa-thologists. Royal Berkshire Hospital, Reading,United Kingdom.

11. Blankenhorn, D. H., and E. H. Ahrens, Jr. 1955.Extraction, isolation and identification of hydrolytic

products of triglyceride digestion in man. J. Biol.Chem. 212: 69.

12. Masarei, J., and W. J. Simmonds. 1966. Fat absorp-tion in pancreatic deficiency in rats. Gut. 7: 114.

13. Dean, R. B., and J. R. Vinograd. 1942. Diffusion ofsolubilized dyes in water and through membranes.J. Phys. Chem. 46: 1091.

14. Hogben, C. A. M. 1966. Fat absorption: a transportproblem. Gastroenterology. 50: 51.

15. Morgan, R. G. H. 1964. The effect of bile salts onthe lymphatic absorption by the unanesthetized rat ofintraduodenally infused lipids. Quart. J. Exptl.Physiol. 49: 457.

16. Davson, H., and J. F. Danielli. 1952. The permeabilityof natural membranes. Cambridge University Press,London. 2nd edition. 123.

17. Borja, C. R., G. V. Vahouny, and C. R. Treadwell.1964. Role of bile and pancreatic juice in cholesterolabsorption and esterification. Am. J. Physiol. 206: 223.

18. Saunders, D. R., and A. M. Dawson. 1963. The ab-sorption of oleic acid in the bile fistula rat. Gut.4: 254.

19. Baxter, J. H. 1966. Origin and characteristics of en-dogenous lipid in thoracic duct lymph in rat. J. LipidRes. 7: 158.

20. Shrivastava, B. K., T. G. Redgrave, and W. J. Sim-monds. 1967. The source of endogenous lipid in thethoracic duct lymph of fasting rats. Quart. J. Exptl.Physiol. 52: 305.

21. Karmen, A., M. Whyte, and D. S. Goodman. 1963.Fatty acid esterification and chylomicron formationduring fat absorption. I. Triglycerides and cholesterolesters. J. Lipid Res. 4: 312.

22. Savary, P., and M. J. Constantin. 1966. Sur la resorp-tion intestinale des chaines eruciques et leur incorpo-ration dans les chylomicrons lymphatiques du rat.Biochim. Biophys. Acta. 125: 118.


absorption of oleic and palmitic emulsions micellar solutions

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