I TRE A~~~R~~~~ OF OLEICACID FROM EMULSION AND MICELLAR SOLUTION I, The absorption of oleic acid by rat jejunum was measured is viva by memur- ing uptake, ~&Qr~ratio~ into ~igly&eride and ti-ansport out of the intestine by tied loops of je$nnm and Z+5 s&2 by meas~i~ its i~~or~r~tion into t~g~y~erid~ by je- junal slices. T&e oleic a& Was dissolved at high ~o~~en~a~~~~ in P=J mM sodium tauro- cholate to compare abso~~~~n from solutions in which mo& was in emulsion @H 5.8) with abs~~~~on from sol~ti~~~ in which most was in micelIar solution fpH ?.a). 2, No difference could be detected between the ~bso~~on from emulsions and ~bs~~~~o~ from micellar sohrtion. Studies were ma& over a vi&Se x-zmge of lipid con- &e~t~tio~ and? is ztk@,over a wide period of time, Abso~~on was related only to total ofdc acid concentration, regardless of its physical form. 3” The in vitro technique was found to be a misleading indication of behaviour in viva, for maximum esterification was achieved at concentrations below complete saturation of the micellar phase, whereas a;n tieto uptake and esterification were directly related to oleic acid ~~~~en~a~ion in the lamen t~~~~ghu~t the range studied, no matter wbetber In emulsion or m.i*Bar sol&5on, 4. It is concluded that absorption carr be as efkient from an emulsion as from a mice&r solution and that oleic acid can be absorbed ~r~~~~ from emulsion droplets without passing through ~axh i~te~edi~ry micellar phase, The micellar phase of intestinal contents is often sited as the phase from which lipid is most efficiently absorbed This, however, has been questioned by SXMM~NS et; 1% who showed that iyrn~h~ti~ adoration of radioactive fatty acid was the 5;hme Erorn an ern~lsi~~ iz~ bile salt sol&ion below cri%iea.l mieellar ~o~~~~a~o~ as from 8 micellar bile salt solution. We have compared the rates of absorption from emulsions with those from mice&r solutions of the same total oleie acid ~o~~~tratio~ ting solutions of &fIerent pH. pH alters the dis#~b~t~~n of oleic acid between micelIar and emulsioir phase+ but
Transcript
I
TRE A~~~R~~~~ OF OLEIC ACID FROM EMULSION AND
MICELLAR SOLUTION
I, The absorption of oleic acid by rat jejunum was measured is viva by memur- ing uptake, ~&Qr~ratio~ into ~igly&eride and ti-ansport out of the intestine by tied loops of je$nnm and Z+5 s&2 by meas~i~ its i~~or~r~tion into t~g~y~erid~ by je- junal slices. T&e oleic a& Was dissolved at high ~o~~en~a~~~~ in P=J mM sodium tauro- cholate to compare abso~~~~n from solutions in which mo& was in emulsion @H 5.8) with abs~~~~on from sol~ti~~~ in which most was in micelIar solution fpH ?.a).
2, No difference could be detected between the ~bso~~on from emulsions and ~bs~~~~o~ from micellar sohrtion. Studies were ma& over a vi&Se x-zmge of lipid con- &e~t~tio~ and? is ztk@, over a wide period of time, Abso~~on was related only to total ofdc acid concentration, regardless of its physical form.
3” The in vitro technique was found to be a misleading indication of behaviour in viva, for maximum esterification was achieved at concentrations below complete saturation of the micellar phase, whereas a;n tieto uptake and esterification were directly related to oleic acid ~~~~en~a~ion in the lamen t~~~~ghu~t the range studied, no matter wbetber In emulsion or m.i*Bar sol&5on,
4. It is concluded that absorption carr be as efkient from an emulsion as from a mice&r solution and that oleic acid can be absorbed ~r~~~~ from emulsion droplets without passing through ~axh i~te~edi~ry micellar phase,
The micellar phase of intestinal contents is often sited as the phase from which lipid is most efficiently absorbed This, however, has been questioned by SXMM~NS et; 1% who showed that iyrn~h~ti~ adoration of radioactive fatty acid was the 5;hme Erorn an ern~lsi~~ iz~ bile salt sol&ion below cri%iea.l mieellar ~o~~~~a~o~ as from 8 micellar bile salt solution.
We have compared the rates of absorption from emulsions with those from mice&r solutions of the same total oleie acid ~o~~~tratio~ ting solutions of &fIerent pH. pH alters the dis#~b~t~~n of oleic acid between micelIar and emulsioir phase+ but
2 J. D. HAMILTON
as demonstrated by WEBB et a1.2, and confirmed here, does not itself alter uptake or esterification from micellar solutions. Bile salt concentration was kept constant, so
avoiding the variable due to the effect of bile salts on esterification2$‘. Absorption was studied in vivo by incubating rat jejunal slices in lipid solution
and measuring incorporation of [l%]oleic acid into triglyceride. In studies in vivo lipid solutions were placed in the lumen of jejunal loops in rats and uptake out of the
lumen, esterification in the mucosa, and onward transport out of the loop were mea- sured over a range of time and of oleic acid concentration to see if a micellar phase is a
prerequisite for optimal obsorption. The in z&o method was assessed to see if it was a valid indicator of rate determining steps in viva at concentrations of oleic acid high
enough to produce an emulsion in 15 mM sodium taurocholate.
MATERIALS AND METHODS
Uniformly r4C-labeled oleic acid was purchased from the Amersham, Searle Corp. and radioactive purity shown to be greater than 96%. Oleic acid (stated purity 99%) was purchased from the Fatty Acid Project, the Hormel Institute, Minnesota, and purity checked by thin-layer chromatography using Silica gel and a solvent solu- tion of hexane-ether-glacial acetic acid (20 : 2 : 0.1, by vol.) Sodium taurocholate was
purchased from Mann Research Laboratories, N.Y. with a stated purity of 97.2%. Male Sprague-Hawley rats, weight 150-200 g were starved over-night before
use.
Radioactive lipid was dissolved in solutions containing 15 mM sodium tauro- cholate, NaCl, KC1 and sodium phosphate buffer (60 mM) to give a final concentration of 135 mM Na+ and 15 mM K+ and a pH of 5.8 or 7.2. Glucose was added to a concen- tration of x or 4 mg/ml. Immediately before use in vitro all solutions were gassed with CO,-0, (I : 20, by vol.) and sonicated for 2 min in a Raytheon sonic oscillator. Emul- sions were visually stable for at least 40 min. The largest Millipore filter through which a clear micellar solution could be obtained by filtration had a pore diameter of IOO pm.
Incubation expeyi~ents in vitro Everted jejunal segments were prepared, weighed, and incubated at 37’ as
described previously4. Contents of the flasks were emptied onto glass wool in filter funnels but not rinsed. The segments were at once transferred to z ml chloroform- methanol (2: I, by vol.) for the extraction of lipids. At the end of 20 min incubation, the PO, of the incubation fluid was not below IOO mm Hg and pH had not changed. Histological sections showed only minor loss of epithelial cells from the tips of villi. Experiments done at different times and with different batches of rats gave compara- ble results within the experiment but mean values for the same experimental variable sometimes differed. To avoid errors due to this, each experiment was constructed as follows. To study the effect of changing a single variable, 6 rats were used, and tissue from each rat was incubated in duplicate for each condition of the variable under study. Statistical significance was evaluated by Student’s t test.
Riochim Bfophys. Adz, 239 (1971) 1-8
ABSORPTION OF OLEIC ACID 3
Experiments in vivo
Under ether anaesthesia two adjacent IO-cm loops of jejunum were tied off with a fine polythene canulla in each. The loops were replaced in the abdomen and into
each was placed 0.25 ml of lipid solution with pH 5.8 in one and pH 7.2 in the other. After a set time the loops were rapidly removed and their contents rinsed out with 5 ml of ether and collected. The tissue of the loops were placed in 5 ml of chloroform-
methanol. The lipids of contents and mucosa were extracted separately so that uptake from the contents, esterification, and transport out of the, loop could be measured. For ‘zero’ time experiments, loops were removed from the rats before injecting lipid solutions and these were washed out of the loop immediately. Each experiment was performed using 4, 6, or IO rats, solutions of each pH being placed alternately in proximal and distal loops. The pH difference of lumen fluid was maintained during
the 3o-min period, changing from pH 5.8 and 7.2 to pH 6.3 and 6.8.
Extraction of lipids Jejunal slices and segments : Tissue was homogenized in chloroform-methanol
and after standing for 30 min was filtered, and one-fifth of the volume of water added to give 2 phases. After centrifugation, the chloroform phase was removed, evaporated to dryness and taken up in hexane. One aliquot was taken to measure radioactivity and another for thin-layer chromatography to separate the triglyceride and fatty
acid fractions. Radioactive lipid in jejunal segments was expressed as nmoles/g of wet tissue. Extraction efficiency for [Wloleic acid by this method was 87%, and was unaffected by tissue weight.
Lumen contents were extracted, after evaporating the ether, by hydrolysing for 8 h at 60” in 15 ml methanol and 15 ml of 30% KOH, acidifying with cont. HCl and
extracting into 40 ml of hexane. Extraction efficiency was 92%. Radioactivity was determined using a Nuclear Chicago scintillation counter.
RESULTS
In vivo experiments (Tables I, II and III, Fig. r) To study the time course of uptake, esterification, and transport out of the
jejunum in vivo, 0.25 ml each of sonicated lipid solution in 15 mM sodium taurocholate containing 2.4 mg/ml of [14C]oleic acid at pH 5.8 and 7.2 were placed in jejunal loops as described and the loops were washed out at ‘zero’ time and at IO and 60 set, 5 and 30 minutes. Table I and Fig. I shows that there was rapid initial uptake of oleic acid
into the mucosa, with a slower incorporation into triglyceride and disappearance out of the loop. At no time was there any difference between any of .these parameters of absorption between solutions of different pH, except for the unesterified fatty acid content of mucosa at 5 min (P = 0.05). At ‘zero’ time there was already uptake of a considerable amount of free fatty acid and at IO set incorporation into triglyceride. There was continuing removal of oleic acid from the lumen contents throughout the 30 min and a continuing incorporation into triglyceride, and diminution in mucosal free fatty acid content so that total mucosal radioactivity, after increasing to 60 set, remained constant. The total absorption out of the loop was rapid in the first 60 set but there-after was very slow.
To study the effect of [Wloleic acid concentration on uptake, esterification and
Biochim. Biophys. Acta, 239 (1971) 1-8
4 100
50 60 . Total Recovery
40
20 : a
0” 0
S 20
40
60
50 i---l FFA
J. D. HAMILTON
0 10 see 1 Mln 5 Min 30 Min
Fig. I. Absorption of oleic acid from jejunal loops in viva. (Retails: set Table I.). TG, triglycerides: FFA, free fatty acids.
TABLE I THE TIME-COURSE OF ABSORPTION OF OLEIC ACID FROM JEJUNAL LOOPS ifi Vi?,0
Two adjacent ro-cm jejunal loops were prepared in each rat under ether anaesthesia leaving a cannula in place for injection into the lumen. After replacement of the loops in the abdomen, 0.25 ml of a sonicated solution of 2.4 mg/ml uniformly i*C-Iabelled deic acid in 15 mM sodium taurocholate was injected into each Ioop, one at pH 5.8 and one at pH 7.2, alternating each between proximal and distal loops in successive rats. At the stated times the loops were removed and the contents immediately washed out with 5 ml of ether. Lipids were extracted from the contents and the tissue of the loops. Tissue lipids were separated by thin-layer chromatography and radio- activity measured for total lipids and free fatty acids and triglycerides. For “zero time” measurements loops were constructed in situ but removed immediately before injecting the lipid solution which in turn was immediately washed out. Results are expressed. as mean i: S.E. percentage recovery of the original dose, as determined by direct extra&ion of a duplicate dose. n refers to the number of rats at each time period. Pi refers to the difference between results from solutions of different pH at the same time period and P refers to the differences between means of pooled results at successive time periods and also between IO set and 30 min. ~.I-._~_ -.- -- _. Ti?VZf n pH [U-14C]Oleic acid (74 of oviginak dose) ~_~._ _-_ ~____ . ..--..-. ~. .._... -
iZlucosa Contents _- -. ~ ~.-. Total Pecouew Total Triglyceride Fatty acid
onward transport in jejunal loops in viva the same preparation was used, and solutions at pH 58 and 7.2 were placed in the loops for 60 sec. [14C]Oleic acid concentration of 0.025~0.5 and 2.5 mg/ml were used (Table II). A direct but non-linear relationship was found at all concentrations between all the parameters measured and the initial con- centration of oleic acid.
There is no significant difference between results for the two pH values except for a minor difference at the highest oleic acid concentration. This, however, is in the opposite direction to those of the means in the previous experiment (Table I), and pooling the results of both experiments for this concentration shows that there is no significant difference.
In a single experiment (Table III) the relationship of uptake, esterification and onward transport to initial oleic acid concentration was maintained when the micellar phase from the two emulsions was used and absorption studied at 2,5, and IO min.
TABLE III
THE INCORPORATION an viva 0F OLEIC ACID INTO TRIGLYCERIDE FROM THE FILTERED MICELLAR
PHASE OF EMULSIONS
The experiments were carried out as described for Table I, and using the filtered micellar phase of emulsions of oleic acid in 15 mM sodium taurocholate at pH 5.8 or 7.2. Each result represents one rat, and is expressed as nmoles of [U-W]oleic acid. --... .-- ----~~~.____ TimA? PfJ DO% [U-W]Oleic acid (nmoles) (min) (laWZOl8S) kucosa
THEEFFECTOFOLEICACIDCONCENTRATION ON lNCORPORATIONlNTOTRlGLYC~RlDE~~ Viti’O
Each incubation flask contained three slices of rat jejunum in 4 ml of 15 mM sodium taurocholate in phosphate buffer at pH 5.8 or 7.2 with the required concentration of uniformly W-labelled oleic acid. All solutions were sonicated before use. After 20 min incubation at 37” in 5ojo CO, in O,, slices were removed and lipids extracted and radioactivity in triglycerides was measured. Results represent the mean & S.E. of duplicate pairs of incubations from 6 rats and are expressed as nmoles of [%]oleic acid incorporated into triglyceride per g wet weight of tissue. P refers to differences between results from different concentrations of oleic acid. P1 refers to differences between results at different pH but the same concentration of olric acid. -I-,_-- --..... ____ ._. -__ [U-W]OEeic acid (mglml) Triglyceride (nmdss [U-W]oleic acid pev g)
pH .pY P pH 7.2 P P’ _.___- ._.. -_-.-_- -_____ _I_
In vitro experiments (Table IV} Jejunal slices were incubated for 20 min in 15 mM sodium ~uro~holate with pH
5.8 or 7.2 and with four concentrations of oleic acid. Table II shows that there was a
significant increase in incorporation into triglyceride as oleic acid concentration rose from 0.025 to 0.075 to 0.5 mg/ml (P -c 0.005) but no significant change between 0.5 and 2.5 mg/m.l. This was found for solutions of both pH and there were no significant
differences between the results at pH 5.8 and 7.2 for concentrations of 0.025 and 0.075 mg~ml (P > 0.05) and barely significant differences between 0.5 and 2.5 mglml (P = 0.03 and 0.04).
Additional studies showed no difference in esterification when glucose coneen-
tration was increased 4-fold. At both the highest and lowest oleic acid concentration esterification continued throughout the zo-min incubation period.
DISCUSSION
Solutions of the same oleic acid concentration but differing physical states have
been prepared by using buffers of differing pH. In this and previous studies2 it has been demonstrated that within the range used here, pH alone does not affect rates of esterification. Bile salt concentration, which is known to affect ester&cation6 has been
kept constant. Studies in vivo demonstrate that the rate of mucosal uptake, incorporation into
triglyceride and transport out of the jejunum are related to the total oleic acid con- centration in solution, regardless of whether most of this is in emulsion or all of it is in micellar solution. When the droplets are removed from an emulsion by filtration (Table III) then absorption is related to micellar concentration. Th~interpretation of these observations is that oleic acid can be absorbed directly from emulsion droplets and it does not need to pass through the intermediary micellar phase. Were this intermediate phase necessary, then absorption would be related only to micellar concentration and not to total concentration. The implication is that the emulsifying properties of bile salts may be as important as their ability to form a micellar solution.
The observations i% vitro are similar to those of CLARK et aLa using palmitic acid
which is solid at 37O. The concentration of oleic acid above which there is no increase in incorporation into triglyceride is below that required to create an emulsion at pH 5.8 or 7.2. This explains unreported personal observations that rates of esterification from emulsions were identical to those from their filtered micellar phases. Since the behavi- our i?z vitro does not reflect the behaviour in vivo then the irt vitro technique does not appear to be a valid tool for the study of absorption from emulsions in bile salt solu- tions of the concentration used here.
ACKNOWLEDGEMENT
This study was supported by The Medical Research Council of Canada. (Grant No. MA 3517). The technical assistance of R. Jodhan and R. Haber is gratefully acknowledged.
8 J. D. HAMILTON
REFERENCES
I W. J. SIMONS, T. G. REDCRAVE AND R. L. S. WILLIX, J. C&z. Invest., 47 (1968) 1015. z J. P. W. WEBB, J. D. HAMILTON AND A. M. DAWSON, Biochim. Biophys. Acta, 187 (1969) 42. 3 A. M. DAWSON AND K. J. ISSELBACHER, J. CL&z. Invest., 39 (1960) 730. 4 J. D. HAMILTON, J. P. W. WEBB AND A. M. DAWSON, B&him. Biophys. Acta, 176 (1969) 27. 5 N. E. HOFFMAN, Biochim. Biophys. Acta, 196 (1970) 193. 6 M. L. CLARK, H. C. LANZ AND J. R. SENIOR, 1. Clin. Invest., 48 (1969) 1587.
