BIOCHIMICA ET BIOPHISICA ACTA 27 BBA 55521 THE ABSORPTION OF TRISTEARIN AND STEARIC ACID AND TRIPALMITIN AND PALMITIC ACID STUDIES ON THE RATE-LIMITING STEPS IN RATS J. D. HAMILTON, J. P. W. WEBB AND A. M. DAWSON St. Bartholomew’s Hospital, London. ECr (Great Britain) (Received August and, 1968) SUMMARY I. In free feeding experiments in rats the absorption of labelled tristearin was poor. It remained constant over a wide dose range, but fell with a high dose. The absorption in bile fistula rats was diminished. 2. The addition of triolein increased the absorption of labelled tristearin in control rats but not in bile fistula rats, suggesting that poor solubility in bile salt solution is an important step hindering the absorption of tristearin and that triolein stimulates absorption by increasing this solubility. 3. Jejunal segments incubated in a micellar solution of equal concentration of labelled stearic acid and oleic acid in IO mM sodium taurocholate took up stearic acid at a slightly faster rate than oleic in a ratio of 0.84, but incorporation into triglyceride was greater for oleic acid in the ratio of 1.3. 4. When a micellar solution of equal concentration of labelled oleic and stearic acids were infused into the duodenum the content of oleic acid in lymph triglyceride was greater than that of stearic acid in a ratio of 1.35. 5. When equal weights of labelled oleic and stearic acids were fed, mixed with bran, the content of oleic acid in lymph triglyceride was much greater than that for stearic acid, in a ratio of 8.9 in the first hour, falling to 4.5 by the fourth hour. These ratios are much greater than those during lymphatic absorption from equimolar micellar solution. 6. We conclude that micellar solubilisation is a major rate-limiting step in the absorption of tristearin and stearic acid, that mucosal cell uptake is not rate-limiting and that esterification is only a minor rate-determining step. 7. The absorption of palmitic acid by control rats was slightly increased by a large dose of triolein added to the feed, but that of tripelmitin was unaltered. Reasons for the difference in behaviour between tripalmitin and tristearin are discussed. Biochim. Biophys. Ada, 176 (1969) 27-36
Transcript
BIOCHIMICA ET BIOPHISICA ACTA 27
BBA 55521
THE ABSORPTION OF TRISTEARIN AND STEARIC ACID AND
TRIPALMITIN AND PALMITIC ACID
STUDIES ON THE RATE-LIMITING STEPS IN RATS
J. D. HAMILTON, J. P. W. WEBB AND A. M. DAWSON
St. Bartholomew’s Hospital, London. ECr (Great Britain)
(Received August and, 1968)
SUMMARY
I. In free feeding experiments in rats the absorption of labelled tristearin was poor. It remained constant over a wide dose range, but fell with a high dose. The absorption in bile fistula rats was diminished.
2. The addition of triolein increased the absorption of labelled tristearin in control rats but not in bile fistula rats, suggesting that poor solubility in bile salt solution is an important step hindering the absorption of tristearin and that triolein stimulates absorption by increasing this solubility.
3. Jejunal segments incubated in a micellar solution of equal concentration of labelled stearic acid and oleic acid in IO mM sodium taurocholate took up stearic acid at a slightly faster rate than oleic in a ratio of 0.84, but incorporation into triglyceride was greater for oleic acid in the ratio of 1.3.
4. When a micellar solution of equal concentration of labelled oleic and stearic acids were infused into the duodenum the content of oleic acid in lymph triglyceride was greater than that of stearic acid in a ratio of 1.35.
5. When equal weights of labelled oleic and stearic acids were fed, mixed with bran, the content of oleic acid in lymph triglyceride was much greater than that for stearic acid, in a ratio of 8.9 in the first hour, falling to 4.5 by the fourth hour. These ratios are much greater than those during lymphatic absorption from equimolar micellar solution.
6. We conclude that micellar solubilisation is a major rate-limiting step in the absorption of tristearin and stearic acid, that mucosal cell uptake is not rate-limiting and that esterification is only a minor rate-determining step.
7. The absorption of palmitic acid by control rats was slightly increased by a large dose of triolein added to the feed, but that of tripelmitin was unaltered. Reasons for the difference in behaviour between tripalmitin and tristearin are discussed.
Biochim. Biophys. Ada, 176 (1969) 27-36
28 J. D. HAMILTON, J. P. W. WEBB, A. M. DAWSON
INTRODUCTION
The poor absorption of stearic acid and tristearin as compared with other lipids has been explained on the basis of their high melting point1 while the improved absorp- tion as a result of mixing with trioleinZ or oleic acid3 was a result of the lowering of this melting point. An alternative explanation was the inefficient uptake* and esteri- ficationb of stearic acid as shown in experiments in vitro. The realisation that fatty acids are probably absorbed from mixed micelles of bile salt, fatty acid and monoglyceride in the intestinal lumen6 has provided a third explanation in that stearic acid alone has a poor solubility in bile salt solution, but that this is greatly improved by the addition of oleic acid and/or mono-olein 6. In the present investigation these possibilities have been examined in an attempt to determine the rate-limiting stages in stearic acid absorption. Some of these studies have been extended to tripalmitin and palmitic acid, which are absorbed better than tristearin and stearic acid, but not as well as triolein and oleic acid.
MATERIALS AND METHODS
From the Radiochemical Centre, Amersham, Bucks, were purchased [I-X]- tristearin, [I-14C]triolein, uniformly 14C-labelled oleic acid, [I-lX]stearic acid and [9,Io-3H,]stearic acid, [I-Xlpalmitic acid and [r-14C]tripalmitin. These were shown to be of greater than 99% radiochemical purity on thin-layer chromatography. Cor- responding unlabelled lipids were purchased from the Hormel Institute.
Sodium taurocholate was prepared according to NORMAN’ and contained less than 0.5% of free cholic acid.
Male rats of Wistar strain, weighing 200-250 g were used.
Lipid for free feeding e+eriments Lipid was incorporated in a pellet of crushed bran from which fat had pre-
viously been removed by ether reflux. Radioactive lipid in doses of 25-100 mg were weighed onto a watchglass and melted into 200 mg oi bran, which was then worked into a pellet with water. For feeds of 200 mg of lipid a larger weight of bran, 300 mg, was needed to take up the whole dose. Feeds of 0.1 mg lipid were added to the bran in hexane, which was allowed to evaporate.
Micellar solutions of fatty acids for incubatiorz in. vitro and duodenal hafusion Radioactive lipid was transferred in hexane to a flask, the hexane allowed to
evaporate; bile salt solution was then added and shaken at 37’ overnight to produce a water clear micellar solution with the following composition : IO mM sodium taurocho- late, 65 mM sodium phosphate buffer, adjusted to pH 7, 60 mM NaCl, 15 mM KCl, I mg/ml glucose, 0.025 mg/ml [9,10-SH,]stearic acid (0.880 mM), 0.025mg/mluniform- ly 14C-labelled oleic acid (0.886 mM). The concentration of the two fatty acids can be taken to be equimolar within the accuracy of the method used in these experiments.
Animal $reparations Rats were starved overnight so that their stomachs were empty at laparotomy.
Operations were performed under ether anaesthesia and experiments started 6-12 h after recovery.
Biochim. Biophys. Ada, 176 (1969) 27-36
ABSORPTION OF TRISTEARIN AND STEARIC ACID 29
Free feeding experiments Unrestrained animals. Unrestrained animals comprised a bile fistula group in
which the bile duct was cannulated above the pancreas with polythene tubing which was led out to drain into a glass pannier sutured onto the back after the technique of VAN ZvE8. For a control group a laparotomy was performed and the duodenum mo- bilised and a pannier sewn onto the back.
Rats were kept on a grid (to prevent coprophagy) in individual cages, without restraint, and allowed free access to a solution of 5% glucose, 0.9”/0 NaCl, 0.04O/~ KCI. Food pellets containing the test lipid were eaten spontaneously from a watchglass and subsequently any uneaten food extracted for determination of radioactivity to cal- culate the exact amount eaten. Faeces were collected on plastic sheeting beneath the grid. After 16 h the rats were killed with ether and the lipid of the stomach, small gut, colon and feaces, each with their contents was extracted separately. Rats were dis- carded if, at post mortem, there was evidence of peritoneal damage or if bile had not drained, or if, on subsequent analysis, it was found that less than 80% of the initial feed had left the stomach.
Restrained aGnats with lymphfistula. The thoracic duct was cannulated by the method of BOLLMAN, CAIN AND GRINDLAY~, modified as described by GALLAGHER, WEBB AND DAWSON~~, and the rats held in a restraining cage. Food was eaten spon- taneously, andlymph collected for hourly intervals into heparinized tubes standing in ice. After 7 h, the rats were killed and the gut removed for digestion and saponifica- tion as described below. Rats were not used if lymph flow was less than 2 ml/h, or if a leak of lymph was discovered at post mortem.
It appears that in the strain of rats used the lymph collection by this technique is not complete, for when [14C]cholesterol, which is absorbed only through thelymphll, was fed in separate experiments, part of that absorbed could not be recoveredin the lymph, and of this some was recovered from the liver. Therefore data on relative rates of lymph transport are presented, and no conclusions can be drawn about total lymph transport. It is known that anatomical lymphatico-venous anastomoses are present in some species that can allow lymph to by-pass a cannula in the thoracic duct and that there are considerable differences in the size of this by-pass, both between species and between strains of species12.
Infusion experiments in lymph jistula rats The thoracic duct was cannulated as described, and the duodenum cannulated
with fine polythene tubing through a duodenostomy. To minimise alterations in the bile salt concentration of the infused solution by endogenous bile and by the large volume of water that lymph fistula rats were found to drink, a bile fistula was made and drinking water withheld. Instead, hydration was maintained by an infusion of o.g% NaCl through the femoral vein. The rat was placed in a restraining cage and warmed to maintain body temperature at about 37”. The micellar solution was infused from a syringe into the duodenum, 0.3 ml every 15 min for 3 h. The syringe was kept at 37” and weighed before and after infusion to measure the total dosegiven, which was close to 0.1 mg of each fatty acid. Lymph was collected hourly for 4 h from the start of the infusion. In two rats collections were continued for 7 h, but the amount of radioactivity in the lymph after 4 h was very small. Criteria for including animals and
Biochim. Biophys. Acta, 176 (1969) 27-36
30 J. D. HAMILTON, J. P. W. WEBB, A.M.DAWSON
methods of lipid extraction from samples were as described for the free feeding expe- riments.
In vitro incubation experiments Jejunal segments were prepared from a rat, starved overnight. The jejunum
was opened longditudinally, and three segments of 0.7 cm, each from a separate third of the jejunum, were placed in watchglasses containing phosphate buffer at pH 7. These were then blotted and weighed, and transferred to incubation flasks, each con- taining 4 ml of micellar lipid solution (see above), which had been previously equili- brated with 5 “/o CO, in oxygen. Incubation was carried out in a Dubnoff shaker at 37” in an atmosphere of 5% CO, in oxygen. In some experiments 46 mg NaF (0.275 M) was added as a metabolic inhibitor-this had previously been shown to reduce incorporation of radioactive fatty acid into triglyceride to less than IO/,.
After incubation, the contents of the flasks were emptied onto glass wool in separate filter funnels and then rinsed twice with 3 ml of IO mM sodium taurocholate. They were then transferred at once to IO ml of chloroform-methanol (2 : I, by vol.) for extraction of tissue lipid. ‘liptake and esterification was studied over a zo-min period. Incubations were in duplicate and each pair was with segments from a different rat.
Extraction of lipids
Gut and faeces. Saponification was completed overnight in 30 ml each of 30% KOH and 309/~ methanol at 60”. After acidification with HCl, lipid was extracted with 50 ml hexane and I ml of the hexane phase taken to determine radioactivity. Absorp- tion was calculated from the difference between what had left the stomach and what was recovered from the gut and faeces, and expressed as percent absorption of what had entered the small intestine. The radioactivity in the small intestine with contents was usually less than 2% and never more than 7% of the radioactivity in the colon and faeces, so the error introduced by counting small intestinal mucosal lipid as being unabsorbed was very small.
Lymph. Lipid was extracted by the method described by SAUNDERS AND DAW-
SON13, and taken up into hexane. One aliquot was taken to measure radioactivity and another taken to separate triglyceride and fatty acid fraction by thin-layer chromato- graphy. These fractions were eluted with diethyl ether. Results were calculated as mg of each radioactive fatty acid in these two fractions in each hour’s collection, and expressed as the ratio between oleic acid and stearic acid. From lymph, as from je- junal segments (below) the radioactivity of total esterified fatty acid was calculated by difference between total radioactivity and that in free fatty acid.
Jejunal segments. Tissue was homogenised in chloroform-methanol and after standing for 30 min, was filtered, and one-fifth of the volume of water added to create two phases. After centrifugation, the chloroform phase was removed, evaporated under nitrogen and taken up in hexane. Total radioactivity and radioactivity in the fatty acid and triglyceride fractions were measured as described for lymph. Results were calculated as mg of each radioactive fatty acid in these two fractions per g wet weight of tissue, and expressed as the ratio between oleic acid and stearic acid. The efficiency of rinsing of the segments was checked in separate experiments in which, after the usual initial rinsing, segments were transferred to a fresh filter funnel and
Biochim. Biophys. Ada, 176 (1969) 27-36
ABSORPTION OF TRISTEARIN AND STEARIC ACID 31
rinsed again by the same technique. In this second rinsing, 10% of total radioactivity for both acids was removed, indicating that the rinsing method was not preferentially eluting one or other acid.
Radioactivity of lipid was determined using a Beckman liquid scintillation system. There was no significant quenching by the samples used.
The significance of results was calculated using Students t test.
RESULTS
Free feeding experiments
The absorption of [I-W]tristearin and the effect of added triolein. In free feeding experiments tristearin absorption was poor at all doses investigated (Table I). No
TABLE I
ABSORPTION OF TRISTEARIN IN CONTROL AND BILE FISTULA RATS
Rats were prepared with either an external bile fistula or a sham operation (control group) and allowed to recover for 6-12 h. Weighed doses of [I-‘Kltristearin were fed in a pellet of bran. After 16 h the rats were killed and the lipid of the stomach, small gut and colon with faeces, each with their contents, was extracted. Absorption was calculated by difference and expressed as the percentage of the dose that had left the stomach. Rats were only used if 80% or more of the dose had left the stomach. P refers to the difference between the two groups.
Dose fed
(mg)
Control Bile fist&a
Absov@on (%) n P Absorption (y/Q n P (mean * S.E.) (mean h S.E.)
25 33.7 f 3 6 13 * 3.3 5 >o.g >0.9
50 34 & 3.8 7 16 i 5.7 5 0.9
100 30 f3 6 <0.02
200 22.5 & 3.5 6
significant difference was demonstrated between the proportion absorbed from 25-,
zo- and roe-mg doses. On the other hand a significantly lower proportion was absorbed from 200 mg (P <0.02, n = 6). Less tristearin was absorbed by bile fistula rats, in whom there was no significant difference between absorption from 25- and 5o-mg doses.
When triolein was added to 25 mg of tristearin in control rats, 25 mg made no difference to absorption, but 100 mg increased absorption from 33.7% to 59%
70
60
r
Fig. I. The effect of two doses of triolein on the absorption of 25 mg of [I-%]tristearin in control and bile fistula rats. Each point is the mean & S.E. for 7 rats (no triolein) and 6 rats (with triolein).
B&him. Biophys. Acta, 176 (1969) 27-36
32 J. D. HAMILTON, J. P. W. WEBB, A. M. DAWSON
(P = 0.01, n = 7) (Fig. I). In contrast, in bile fistula animals, neither dose of triolein had any effect.
The absorption of [Xlstearic acid. The absorption of 25 mg stearic acid (44.7 f 4, mean & SE.) was not significantly different from that of 25 mg tristearin (33.7 f 3.2, mean & S.E.).
The absorption of [r-W] tviolein. The absorption of 25 mg and zoo mg of triolein, the extremes of the doses of triolein used in mixed feeding experiments, was almost complete (95% and 96%).
TABLE II
THE UPTAKE AND INCORPORATION INTO TRIGLYCERIDE OF OLEIC ACID AND STEARIC ACID BY RAT
JEJUNAL SLICES in ZdVO
Each incubation flask contained three slices of rat jejunum in 4 ml of IO mM sodium taurocholate in phosphate buffer (pH 7) with 0.025 mg/ml each of uniformly i%-labelled oleic acid and [g,~o-3H,]
stearic acid. They were incubated at 37” in 5% CO, in oxygen, and then removed and rinsed and tissue lipids were extracted, and radioactivity measured in free fatty acid and triglyceride fractions. Results are expressed as a ratio of oleic to stearic. Where indicated, 46 mg NaF was added to the flasks as a metabolic inhibitor. n is the number of duplicate pairs of incubations; each from a different rat. P refers to the difference of the ratio from unity.
Incubation Addition n Ratio of uniforml?, 14C-labelled oleic acid/[g,Io_3H,]stearic acid time (mean + S.E.) (min)
.___ Fvee fatty acid Triglyceride Total esterified
Absor;htion from micellar solution In vitro (Table II). At each incubation time up to 20 min, the ratio in the free
fatty acid fraction of oleic to stearic acid tended to favour stearic acid with a mean of 0.84. The same was found in tissue poisoned with fluoride. The ratio in the triglyceride fraction, however, favoured oleic acid at all times, with a mean of 1.3. The poor in- corporation of stearic acid into triglyceride was not offset by a better incorporation into some other fraction such as phospholipid, for the incorporation into total esterified lipid also favoured oleic acid. Of total radioactivity, esterified fatty acid accounted for 72% (stearic) and 78:/, (oleic) and triglyceride accounted for 40% (stearic) and 50% (oleic).
Lymphatic absorption in vivo. Fig. 2 shows the labelled fatty acid content of the triglyceride fraction of hourly lymph collections in a typical experiment. In each of the first 4 h the oleic acid content was greater than the stearic acid content. After 4 h the total radioactivity was very small, and so measurements of the first 4 h are used in Fig. 3, where the relative contents of the two labels in triglyceride in five experiments have been expressed as a ratio of oleic to stearic acid in a manner similar to Table II. The content of oleic acid was always greater than stearic acid, with a mean ratio of 1.35, which is similar to that for incorporation into mucosal triglyceride in vitro.
As with incubations in vitro, the impaired transport of stearic acid was not offset by enhanced incorporation into other fractions, as essentially the same ratio
Biochim. Biophys. Acta, 176 (1969) 27-36
ABSORPTION OF TRISTEARIN AND STEARIC ACID 33
1 HOURLY fmw CLcxd *mm L*m 0F6~~FUS10~7
Fig. 2. The content of uniformly W-labelled oleic acid and [g,ro-SH,]stearic acid in the trigly- ceride fraction of hourly lymph collections after the start of a duodenal infusion of micellar solution of equal concentration of the two fatty acids. Representative experiment.
(1.38) obtained for total esterified fatty acid which made up 98% of the total radio- activity for each fatty acid. Radioactivity of triglyceride was 71% of the total for each fatty acid.
The dose administered in infusion experiments was about 0.1 mg and as this
was very small compared with the z5-mg dose in the free feeding experiments describ- ed above, it was necessary to show that there was a difference in absorption between oleic and stearic acid at the small dose also-otherwise experiments using this dose might be irrelevant in defining the cause of differences demonstrated with the higher dose range. When 0.1 mg each of stearic and oleic acid were fed together there was in- deed a difference in their absorption (controls: steak, 83% and oleic, g5%, P ~0.01, n = 7; bile fistula: stearic 21% and oleic 58%, P <O.OI, n = 7), although the differ- ence was not so great as with larger doses of free acid or triglyceride.
Lymphatic absorption of uniformly “C-labelled oleic acid and [g,Io-3Hz]stearic acid from
feed (Figs. 3 and 4) Finally a study was made of the relative contents of the two labelled acids in
11
0
HU"RLYL:MPH2COL3LECT4,0N:AFRRF7EED WAY LYM3PHCOLL~CTI(INS:FTERFEbED 7
Fig. 3. The ratio of the content of uniformly “C-labelled oleic acid and [g,r+H,]stearic acid in the triglyceride fraction of hourly lymph collections after (I) infusion of micellar solution of total of 0.11 mg of each acid as in Fig. z (mean -& S.E. of 5 experiments) and (2) feeding of 25 mg of each acid as in Fig. 4 (mean f S.E. of 6 experiments).
Fig. 4. The content of uniformly i4C-labelled oleic acid and [g. ro-3H,]stearic acid in the triglyceride fraction of hourly lymph collections after feeding of 25 mg of each acid. Representative experiment.
Biochim. Biophys. Ada, 176 (rghg) 27-36
34 J. D. HAMILTON, J. P. W. WEBB, A. M. DAWSON
lymph triglyceride after feeding 25 mg of each together in the same bran feed. In contrast to the infusion experiments, solubilisation has now to be accomplished during digestion, and Fig. 4, which is the result of a typical experiment, shows that in each hour’s lymph the content of oleic is much greater than that of stearic acid. Fig. 3 de- monstrates that the ratio between them is very much greater than in the infusion ex- periment. Moreover, the difference is greatest in the first hour, and diminishes there- after. In these experiments stomach emptying was not complete at 7 h, and the stomach contained the same amount of each acid, indicating that they entered the intestine at the same rate. Total absorption was 83% for oleic acid and 44% for stearic acid (P <O.OI, n = 6). In the lymph, of total radioactivity, esterified lipid contained 99% (oleic) and 970/L (stearic) and triglyceride contained 76% (oleic) and 687/, (stearic).
TABLE III
THE ABSORPTION OF PALMITIC ACID AND TRIPALMITIN AND THE EFFECT OF ADDED TRIOLEIN
Sham operated rats were fed z5-mg doses of [I-‘%]palmitic acid and [I-14C]tripalmitin in bran pellets, with and without IOO mg or zoo mg of triolein. Absorption was calculated and expressed as a percentage of that which left the stomach as in the legend to Table I. P refers to the difference between the two groups. All groups contained 7 rats.
Added tviozein
(mgl
o/0 Absorption (mean 5 S.E.)
Palmitic acid Tri$alxp
0
100 200
__-
66 & 4* 70 * 5** P = 0.5 P = 0.5
69 i 4 66 + 4 78 + 4* 72 * q**
* P = 0.01 ** P = 0.3.
The absorption of [l-14C]palmitic acid ad [I-W]tripalmitin and the efect of added
triolein (Table III) There was no difference between the absorption of 25 mg palmitic acid (660/,) or
tripalmitin (70%) which was more efficient than that of tristearin. The addition of triolein to tripalmitin made no difference to its absorption either with IOO mg (66%) or 200 mg (720/,) (P = 0.5 and 0.3). With 25 mg palmitic acid, IOO mg of triolein caused no change in absorption (69%) but 200 mg caused a significant increase to 789; (P (0.01).
DISCUSSION
The poor absorption of tristearin is clearly not due to an early saturation of absorptive capacity, for a constant proportion was absorbed over a wide range of doses.
Lipolysis is not rate-limiting, as indicated by the equal absorption of free fatty acid and triglyceride which occurred with both tristearin and tripalmitin.
The effect of triolein on stearic acid absorption is similar to the finding of MATTIL AND HIGGINS~ in rats, and also to that of RENNER AND HILLY and YOUNG AND
GARRETT~~ who found that the absorption of stearic acid by chicks was improved by
Biochim. Biophys. Acta, 176 (1969) 27-36
ABSORPTION OF TRISTEARIN AND STEARIC ACID 35
oleic and linoleic acids. It is unlikely that the stimulation of tristearin absorption by triolein is due to a simple lowering of melting point, for it should then have stimulated absorption in the bile fistula animals. Moreover, the melting point of the mixtures used were above 37’ (unpublished observations) so the triolein could not have caused the formation of a liquid emulsion. The observations suggest that the stimulation is due to an increase in solubility of stearic acid in bile salt micelles, a mechanism which obviously cannot operate in bile fistula animals. We have confirmed the observation in vitro of HOFMANN AND RORGSTR~~M~ that mono-olein and oleic acid, either individ- ually or together, increase the solubility of stearic acid in micellar bile salt solution. The lack of effect with 25 mg of triolein may have been due to the rapidity of triolein absorption noted previously by BORGSTK~M~~ and in our own experiments, so that mono-olein and oleic acid might be removed before they have a detectable effect on
absorption. It is unlikely that mucosal uptake from a micellar solution is a rate-limiting
step, for, in incubation experiments, the uptake of stearic acid was actually greater than oleic acid, both in living mucosa and in mucosa poisoned with fluoride in which there was no incorporation into triglyceride. This observation differs slightly from that of Fox4who, using turtle gut, found that stearic acid was taken up less well than oleic acid. Fox, however, used albumin to solubilise the fatty acids, and the small difference in uptake that she found would not be enough to account for the large differences in absorption found in free feeding experiments.
Incorporation of stearic acid into triglyceride both in the mucosa and in lymph is certainly less than oleic acid, but the difference is far too small to account for the large difference of incorporation of these acids into lymph triglyceride seen in the free feeding experiments. This indicates that the main rate-limiting step in stearic acid and tristearin absorption is the solubilisation of stearic acid in micellar bile salt solu- tion in the lumen. The improvement in absorption might be a reflection not only of static solubility, but also of the rate of solution. This rate might be increased when the melting point of stearic acid is lowered by triolein. When triolein is added to stearic acid in a ratio of 4: I, the melting point is lowered from 69” to 59” (unpublished obser- vations). The design of these particular experiments however does not differentiate between these two aspects of solubilisation.
Our observations are not in agreement with those of GELB AND KESSLER~~ who found a slightly better esterification of stearic acid than oleic acid. They, however, used hamster gut and used albumin to solubilise the fatty acid.
The pattern of lymph transport when the two acids were fed as 25-mg doses in food (Fig. 3) is compatible with a rapid absorption of oleic acid, similar to that de- monstrated with triolein15, and suggested above as a possible reason for the failure of a small dose of triolein to increase the absorption of tristearin. The absorption of palmitic acid is increased to a small extent by 200 mg of triolein, but not by IOO mg. An increase is in agreement with the findings of RENNER AND HILLY and YOUNG AND GARRETT’~. The increase however, is small, and might be a reflection of the observa- tion irk vitro that mono-olein causes a much smaller proportionate increase in micellar solubility of palmitic acid compared with that of stearic acid. HOFMANN AND BORG- STREAMS demonstrated that the solubility of palmitic acid (0.45 mmole/l in 4 mM sodium taurodesoxycholate) was about four times that of stearic acid. The addition of mono-olein in increasing amounts caused a stepwise increase in these solubilities, and
Biochim. Biophys. Ada, 176 (1969) 27-36
36 J. D. HAMILTON, J. I?. W. WEBB, A. M. DAWSON
that this was much more marked for stearic than palmitic, so that with 3 n:moles/l
of mono-olein, their solubilities were fairly close at about I mmole/l. The improve-
ment in palmitic acid absorption is less marked than that observed by YOUNG AND
GARRET-T’~. They, however, were using chicks, and found a much lower absorption of
palmitic acid when this was the only lipid fed.
The failure of triolein to improve the absorption of tripalmitin might be due to
the competition for solubility between mono-olein and fi-monopalmitine, which
would be formed in the gut lumen, so offsetting the improvement in palmitic acid
solubility.
ACKNOWLEDGEMENTS
We wish to thank the Board of Governors of St. Bartholomew’s Hospital and
the Dowager Countess Eleanor Peel Research Fund for support for this work.
REFERENCES
I A. I~. S. CHENG, M. G. MOREHOUSE AND H. J. DUEUL, J. N&r., 37 (1949) 7.37. 2 K. F. MATTIL AND J. W. HIGGINS, J. N&r., 2g (1945) 255. 3 R. RENNER AND F. W. HILL, J. Nut?., 74 (1961) 254. 4 A. IX. Fox, Co++@. Biochem. Physiol., 14 (1965) 553. 5 I. A. HANSEN, Camp. Biochem. Physiol., 15 (1965) 27. 6 A. F. HOFMANN AND B. BORGSTR~~M, Federation Proc., ZI (1962) 43. 7 A. NORMAN, Arkiv Kemi, 8 (1955) 331. 8 A. VAN ZYL, J. Endocrinol., 16 (1957) 213. g J. L. BOLLMAN, J. C. CAIN AND J. H. GRINDLAY, J. Clin. Lab. Med., 33 (x948) 349.
10 N. GALLAGHER, J. WEBB AND A. M. DAWSON, Clin. Sk., 29 (1965) 73. 11 S. A. HYUN, G. V. VAHOUNY AND C. R. TREADWELL, Biochim. Bio$hys. Acta, 137 (1967) 306. 12 A. M. DAWSON, N. GALLAGHER, D. R. SAUNDERS AND J. WEBB, in Ii. M. C. DAWSON AND
D. N. RHODES, Metabolic and Physiological Significance of Lipids, Wiley, London, 1964, p. 243. 13 D. R. SAUNDERS AND A. M. DAWSON, Gut, 4 (1963) 254. 14 R. J. YOUNG AND R. L. GARRETT, J. N&r., 81 (1963) 321. 15 B. BORGSTR~M, PYOC. Nutr. Sot.. 26 (1967) 34. 16 A. M. GELB AND J. I. KESSLER, Am. /. Physiol., 204 (1963) 821.
