Symposium on Fat Absorption

Vol. 50, No.1 Printed in U.S. A .

GASTRO ENTEROLOGY

Copyright

© 1966 by The Williams & Wilkins Co.

SYMPOSIUM ON FAT ABSORPTION* FAT ABSORPTION: A TRANSPORT PROBLEM C. ADRIAN M. HOGBEN, M.D., PH .D. Department of Physiology, UniveTsity of Iowa College of Medicine, Iowa City, Iowa

vironment. Dr. Hofmann subsequently develops this in a much more sophisticated fashion. For the moment, I will assume that the problem of passage across the mucosa is one of accounting for movement out of a micellar solution; that is a saturated solution of fatty acid and monoglyceride whose absolute concentrations are much less than that represented by polymolecular micelles. I am suggesting that today we are closer to knowing from where we start. The ultimate end point, transport away from the mucosa, is the same as it was over a hundred years ago: the appearance of chylomicrons in lacteals. Between these two points, we have to consider three steps in passage across the epithelial barrier: 1. Penetration of the microvillous plasma membrane. 2. Migration through the cell interior. 3. Penetration of the basal plasma membrane. We have been seeking an explanation for the first and perhaps the third of these steps in terms of either microphagocytosis (pinocytosis) or diffusion. You may protest my gratuitous pedantry when I seek to relabel as microphagocytosis what others are calling pinocytosis. But in a strict sense, membrane engulfment, if it plays any important role in normal absorption of fat, does not lead the cell to drink its environment by pinocytosis. Inert solutes such as mannitol or inulin do not pass into the epithelial cell. For the older "particulate hypothesis" that has evolved into microphagocytosis, we should identify the particle. We can use the following rough classification based on particle diameter in Angstroms: chy 10microns 5000 A, microchylons 500 A, micelles 50 A, and molecules 10 A. The size

The gastroenterologist concerned with malabsorption might be perturbed because he has poor perception of events that make possible the absorption of fat. Conflicting views have been expounded and all too frequently the conclusions have been more clearly stated than the questions that should have been asked. It is my hope that this symposium will disclose that the steady progress of research is leading to a consensus. In this introduction, I would like to consider absorption of long chain triglycerides from the point of view of a general physiologist. As one who has been intrigued by the enigma of fat absorption but not actively investigating the problem, I shall endeavor to stress some principles which may sharpen the focus of review. Before reaching a conclusion about any problem of gastrointestinal absorption, we should be prepared to evaluate four factors: (a) movement through the alimentary tract, (b) the physical chemical state of the absorbed material within the lumen, (c) passage across the epithelial barrier, and (d) transport away from the mucosa. While it is true that the first of these, gastrointestinal transit, is highly important in determining the actual extent of absorption, I can, as a general physiologist, with due caution disregard this in discussing mechanisms of transport. It is, however, absolutely essential that we define the physical chemistry of the epithelial en-

* P apers presented at the Symposium of the Gastroenterology Research Group, Annual Meeting of the American Gastroenterological Association, Montreal, Canada, May 27, 1965. Address requests for reprints to: Dr. C. Adrian M. Hogben, Department of Physiology, University of Iowa College of Medicine, Iowa City, Iowa 52241. 51

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HOGBEN

of the particle implicated has shrunk progressively since Frazer first advanced his controversial view.! Electron microscopy provides no support for migration of enormous chylomicrons through the epithelium. I have coined a term, microchylons, to designate intermediate aggregates demonstrated by Palay and Karlin in their electron micrographs of the columnar cel1. 2 In view of the thorough study presented by Dr. Rubin in this symposium, it might be redundant to raise an additional objection to the microphagocytosis of particles as large as or larger than microchy Ions. The intestinal epithelium is lined by a mucous coat. While its function is not known, it is certainly not a diffusion barrier to small solutes such as the hydrogen ion. But gels do form a molecular sieve that can be expected to impede large molecules or aggregates. 3 Though lacking explicit information about the sieve character of the mucous coat, we can suggest that sufficiently rapid absorption of chylomicrons has always been improbable and that of microchylons doubtful. Knowing more now about the physical chemistry of the luminal contents, we are really interested in the fate of the smaller particles, the micelles and molecules. There is no crucial evidence bearing upon the question of a microphagocytosis of micelles. We have to resort to a priori reasoning. A stumbling block to embracing microphagocytosis of micelles is the fate of bile acids. It is now known that the bile acids, which are such an important part of the micelle, are not absorbed (lumen to interstitial fluid) along with the products of triglyceride hydrolysis. For micellar microphagocytosis, it is essential to stipulate that there are pumps in the proximal gut for actively transferring bile acids back into the lumen. This is not impossible, but we would be committing ourselves to an additional ad hoc postulate to preserve our prejudices. No one has yet executed a simple experiment to determine whether the bile acids pass into the epithelial cell by simply exposing the luminal surface to labeled bile acid and a non permeating solute such as mannitol. Failure to demonstrate a significant intracellular concentration would

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not absolutely preclude microphagocytosis but demonstration of intracellular bile acid accumulation would remove a major obstacle to micellar penetration. Thus it is probable that micelles deaggregate before absorption. We then have molecular fatty acid and monoglyceride. It is no longer necessary to evoke microphagocytosis. In spite of the growing acceptance of the concept of the plasma membrane as a lipoid-sieve, there are, as far as I know, no explicit measurements of the rates of permeation of the plasma membrane by extremely lipid soluble solutes. Nevertheless, for the short distance to be traversed, 100 A, I do not know of any conceptual barrier to an extremely rapid permeation of fatty acid by simple diffusion. This is not to belittle the importance of intraluminal micelle formation. The absolute concentration of the saturated solution of fatty acid and monoglyceride is extraordinarily low. Given that these molecules can cross the plasma membrane with ease, provided that there is a "sink" on the farther side, the intraluminal solution would rapidly become desaturated. Without micelle formation diffusion and phase transition could become limiting. I am suggesting that the location of the bile acid micelles is intraluminal and that we may come to regard these as having an important role as mobile "carriers" between emulsion and the plasma membrane. As I shall elaborate shortly, there are other problems in attempting to explain the whole process of fat absorption in terms of simple diffusion. But it is not in the first step of penetrating the plasma membrane. I might parenthetically point out that it is absurd to invoke at this locus a process of active transport in the conventional sense. Not only is it unnecessary, but it is hard to imagine how it could work. To move a solute from a lower to a higher concentration not only requires a special machinery, but it is also necessary that the boundary be relatively impermeable to the solute which is being actively transported. Otherwise, the process would be prohibitively costly to run. For many there is a dichotomy between

January 1966

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FAT ABSORPTION: A TRANSPORT PROBLEM

complete and rapid absorption. I suggest the adjectives are functionally synonymous. Since the sojourn of material in the proximal bowel is relatively short, perhaps less than an hour, absorption must be rapid if it is to be essentially complete. For transfer to proceed rapidly by diffusion, there must be a sufficient starting concentration, the source, and a reduced concentration at a farther point, the sink. Both of these will have to be emphasized. It is now necessary to focus on the second step, migration through the columnar cell. Unlike other circumstances encountered in transepithelial transfer, we are not really dealing with a "membrane" problem at all but with the problems that arise from the very limited solubility of fat in aqueous compartments. No matter how it occurs, it is hard to believe that migration through the cell is not the result of diffusion , and diffusion over a distance of 25 p.. In the case of ions, sugars, or amino acids, we do not ordinarily consider this to be of any consequence and regard the cell interior as essentially a well mixed compartment. But this is not the case for fat transport. To make this point, I am going to burden you with some numerical values. To develop the conclusion, I am going to presume that the metabolic machinery of the cell, by achieving triglyceride synthesis and chylomicron formation at the basal border of the cell, can create the necessary sink. The question will be: once fatty acid has entered the cell can it diffuse through the cell at a sufficient rate to sustain absorption? (Emphasis is placed on the diffusion of fatty acid not only because a larger fraction is absorbed as such, but also because the aqueous solubility of monoglycerides is greater; see Hofmann, this symposium.) I am going to solve the classical Fick equation for diffusion (table 1). The value for the diffusion coefficient for molecularly dispersed fatty acid must certainly be about the value assumed, 5 X 10- 6 cm 2 sec-I. Based on the work of Borgstrom et al.,4 fat absorption is essentially complete in the first third of the small intestine. From the figures calculated by Wilson,5 I arrive at a maximal value of 33,000 cm 2 for the epithe-

TABLE

1. Absorption of fatty acid by diffusion

dn Rate of diffusion: - dt

Diffusion coefficient Surface area Concentration (oleic acid) Distance

D A

=

~c

=

de DA dx

=

=~e

~x

5 X 10- 6 cm 2 sec- 1

= 3.3 X 104 cm 2

3 X 10-2 mg cm- 3

(pH 7.45) ~x =

Q = 2 mg see-

2.5 X 10- 3 cm 1

Normal absorption: 27 g/3 hr =

2.5 mg see-1

lial area involved. I have yet to find any satisfactory studies on the solubilities of fatty acids. The value given here for oleic acid, 0.03 mg cm- a, is taken from the work of Dewitt Goodman 6 on the distribution of fatty acids between saline having a pH of 7.45 and heptane. (In the paper of Goodman cited,6 it was stated that oleic (as well as palmitic and stearic) acid was insoluble just above the highest concentration studied. For oleic acid, the concentration in heptane was 0.063 M with a partition ratio of 900, yielding an aqueous solubility of 0.07 mM or 0.02 mg per cm 3 . The problem of the solubility of fatty acid moieties in an aqueous solution is not simple. Recently, Mukerjee 7 has re-examined the data of Goodman and reached the conclusion that the long chain fatty acids are predominantly present as anion dimers.) Goodman has cautioned me that the oleic acid value may be too low, but I have used this as the best available estimate. I am proceeding on the assumption that the cytoplasm is saturated at the microvillous border and that the concentration is nearly zero at the base of the cell. The concentration falls over a distance of 25 p.. Upon substitution of the above in the Fick equation, the result is that diffusion might sustain transfer at a rate of 2 mg sec-I. I am not yet satisfied that we have measured the maximal capacity to absorb "digested" triglyceride. (Measurement of the apparent maximal absorptive capacity in the rat has yielded values comparable to that cited for man. The earlier work of

HOGBEN

54 FATTY ACID ~DIFFUSION

DIFFUSION

• " " TRIGLYCERIDE a CHYLOMICRON FORMATION

FIG. 1. A diffusion hypothesis for triglyceride absorption (adapted from Wilson").

Deuel et al. 8 indicates a slightly more rapid rate for absorption of coconut oil than determined by Bennett and Simmonds. 9 These studies within their scope set limits, but they may not preclude a more rapid absorption from a prefabricated micellar solution.) The work of Borgstrom, et al. 4 indicates that we can absorb something like 27 g of fat in the course of 3 hr or a rate of 2.5 mg sec- 1 . You might consider that these values are sufficiently similar to leave no problem. However, I have deliberately loaded the dice in favor of diffusion. It should to be pointed out that the available value for the maximal solubility of fatty acid in an aqueous medium is for a pH of 7.45. We usually believe that the pH of the cell interior is significantly less than this and, if the pKa of a fatty acid is 6 or less, the

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solubility would be that much less. The solubility of palmitic and stearic acids would appear to be one-fifth and one-tenth that of oleic acid. Further, it is required that the solution at the microvillous border remain saturated and that at the basal border be maintained close to zero. Consequently, I suggest that when fatty acid has entered the epithelial cell in a molecularly dispersed form, its subsequent diffusion cannot sustain absorption. Then how is absorption accomplished? Do fatty acids reaggregate? This is a matter for speculation. We do not have evidence that bile acids are available intracellularly for the continued maintenance of micelles. Unfortunately, we still do not know whether we should regard the cell interior as a gel or a solution. If it were a gel, it might form a sieve hindering the migration of particles of the dimensions of a micelle or larger. There is the possibility that metabolic conversion of a fatty acid to a more soluble form comes to play an important role in facilitating diffusion. There is also what seems to me the least likely possibility: that the intracellular pH is much higher than elsewhere. The third step is the permeation of the basal plasma membrane. If we are forced to speculate about the fat in the cell interior, we have to be quite hesitant in commenting on the way in which fat leaves the cell. It is not so much a matter of how fat leaves the cell but why. If we could determine that triglyceride formation actually occurred essentially extracellularly at the plasma membrane face in contact with interstitial fluid, it would then remain only to clarify intracellular migration. The sink for diffusion would have been created, and we would not have to invoke the mysticism of microphagocytosis. With some hesitation, I offer a diagram of fat absorption adapted from the monograph of Dr. Thomas Wilson (fig. 1).5 It is possible to entertain the idea that fat enters the epithelial cell as fatty acid by diffusion of the molecularly dispersed form. Subsequent migration requires that fatty acid be metabolized to a more soluble

J anua1'Y 1966

FAT ABSORPTION: A TRANSPORT PROBLEM

form or that it reaggregate. Finally, at the base of the cell, triglyceride synthesis and chylomicron formation establish a sink for the continuous and rapid flow of fat through the epithelial cell. Unlike circumstances that obtain for absorption of other materials, the continuous generation of this sink within the mucosa is a necessary condition. Otherwise, absorption of even a small fraction of fatty acid would saturate the interstitial compartment. To my way of thinking, absorption of fat by diffusion has much merit. But it would not obtain without metabolic transformations. Dr. Isselbacher properly concludes this symposium with a much more cogent commentary on the intestinal metabolism of fat. REFERENCES 1. Frazer, A. C., W. F . R . Pover, H. G. Sammons, and R. Schneider. 1956. Further observations on the absorption of fat, p. 331-340. Interna-

tional Conference on Biochemical Problems of Lipids. 2. Palay, S. L., and L. J. Karlin. 1959. An electron

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microscopic study of the intestinal villus. II. The pathway of fat absorption. J. Biophys. Biochem. Cytol. 5: 373-384. 3. Ackers, G. K., and R. L. Steere. 1962. Restricted diffusion of macromolecules through agar-gel membranes. Biochim. Biophys. Acta 59: 137149.

4. Borgstrom, B., A. Dahlqvist, G. Lundh, and J. Sjovall. 1957. Studies of intestinal digestion and absorption in the human. J. Clin. Invest. 36: 1521-1536. 5. Wilson, T. H. 1962. Intestinal absorption, p. 263.

W. B. Saunders Company, Philadelphia. 6. Goodman, D. S. 1958. The distribution of fatty

acids between n-heptane and aqueous phosphate buffer. J. Amer. Chern. Soc. 80: 38873892. 7. Mukerjee, P. 1965. Dimerization of anions of

long-chain fatty acids in aqueous solutions and the hydrophobic properties of the acids. J. Phys. Chern. 69: 2821-2827. 8. Deuel, H. J., L. Hallman, and A. Leonard. 1940. The comparative rate of absorption of some natural fats. J. Nutr. 20: 215-226. 9. Bennett, S., and W. J . Simmonds. 1962. Absorptive capacity and intestinal motility in unanaesthetized rats during intraduodenal lDfusion of fat. J. Exp. Physiol. 57: 32-38.