epithelium occluded by folding cannot participate in absorptive activity. In

655 J. Physiol. (I955) I30, 655-664 THE ABSORPTION OF WATER AND OF SOME SMALL SOLUTE MOLECULES FROM THE ISOLATED SMALL INTESTINE OF THE RAT By R. B. FISHER From the Department of Biochemistry, University of Oxford (Received 2 June 1955) Wells (1931) showed that the rate of absorption of water from the small intestine of the dog was dependent on the distension pressure. This observation has formed the basis of a view that water absorption is determined by hydrostatic and osmotic factors. The ability of the intestine to absorb the animal's own serum or an isotonic salt solution requires on this view that the absorption of water should be secondary to the absorption of the protein and salts. The surviving small intestine preparation of Fisher & Parsons (1949) provides means of obtaining further evidence of the nature of water absorption, since osmotic conditions on the two sides of the mucosa can be varied at will, -and a useful range of distension pressures can be employed. This paper describes the effects of alteration of distension pressure, osmotic pressure and other factors on the absorption of water and of urea, creatine and sorbitol. In the original work of Wells, in which very low distension pressures were used, some part, if not all, of the effect of distension might be expected to arise from the separation of mucosal folds with increasing pressure: mucosal epithelium occluded by folding cannot participate in absorptive activity. In the present work, therefore, the lowest distension pressure used has been one known, from microscopic examination of preparations fixed during distension, to separate all mucosal folds. A preliminary account of some of this work has appeared in the Proceedings of the Physiological Society (Fisher, 1954). METHODS Setting up and management of the surviving intestine The setting up of the preparation differed only in small details from the description of Fisher & Parsons (1949). The whole small intestine from the duodenal-jejunal junction to the ileo-caecal valve was set up on one circulation unit. The length of such a segment is about 100 cm. All

656 R. B. FISHER experiments reported were on such segments and each lasted for 1 hr. One observation was made on each segment. For the measurement of water and solute absorption two methods were used. In the majority of experiments approximately 75 ml. of Krebs's bicarbonate medium was placed in the inner circuit of each unit. The outer circuit contained approximately 50 ml. of Krebs's medium. As soon as the intestine was set up, a measured volume of Krebs's medium containing a known concentration of urea, creatine or sorbitol was added to the inner circuit contents circulating through the lumen, and well mixed with them over a period of about 2 min. A sample of known volume was then withdrawn. Absorption was allowed to proceed for 1 hr, a second sample of known volume was withdrawn and the remaining fluid in the inner circuit was washed out quantitatively. The initial and final concentrations and the inner circuit content of the solute used as a volume 'indicator' were measured. The change in water and solute content of the inner fluid could be obtained directly from these data. In a few experiments an exactly measured volume of medium was placed in the inner circuit, and the residue collected by drainage at the end of the experiment. Analytical procedures Urea was estimated by hydrolysis with urease and determination of the ammonia after steam distillation from sodium borate buffer. As the intestinal contents often contain sufficient urease activity to cause appreciable hydrolysis of the urea in samples before the analysis could be carried out, the total ammonia content of samples after urease hydrolysis was taken as a measure of the urea present. Creatine was estimated by the diacetyl procedure of Ennor & Stocken (1948). Sorbitol was estimated by titrating the acid liberated on oxidation by sodium metaperiodate. The procedure was the same in principle as that described by Fisher & Lindsay (1955) but 0-3 M-metaperiodate was used instead of 0 03 M, and the titration was performed with N/20-NaOH. Glucose was removed by yeast fermentation before the estimation. RESULTS Glucose concentration and distension pressure as factors determining water absorption Since it had been observed in earlier unpublished work that the presence of glucose in the fluid circulating through the lumen of the surviving small intestine made a considerable difference to the rate of diminution of volume of this fluid, a series of experiments was made to study the effects of glucose concentration and of distension on water absorption. Half the experiments were made with 500 mg/100 ml. of glucose in the inner fluid, and half with no glucose. In all experiments there was 500 mg/100 ml. of glucose in the outer fluid. Half the experiments in each set were made at 20 cm H20 distension pressure and half at 45 cm. The lower pressure was chosen as the least pressure which could safely be supposed to expose all the mucosa. The higher pressure was the highest afforded by the circulation units. The results of the experiments are presented in Table 1. These experiments were performed on male rats, and the volume indicator was urea in all instances. Although the mean rate of water absorption at the higher distension pressure is greater than that at the lower pressure, whether

WATER ABSORPTION 657 or no there was glucose in the inner fluid, the mean of all rates at high distension differs from the mean of all the rates at low distension by little more than the standard error of the difference. Further, in the absence of glucose the rate of water absorption is not significantly different from zero at either distension pressure. TABLE 1. The effects of change in distension pressure and of the presence of glucose in the inner fluid on the rate of water absorption from the isolated rat small intestine Distension Mean rate of water absorption (fl./cm/hr) pressure, A A (cm H20) Glucose present Glucose absent Mean 20 159±43 (4) -12±41 (4) 73±30 (8) 45 213±42 (6) 26±36 (6) 119±28 (12) Mean 191+31 (10) 11±28 (10) Differences 180±41 (20) 46±42 (20) All animals were adult males. Outer fluid contained 500 mg/100 ml. glucose in all experiments. The same glucose concentration was used in inner fluid in experiments labelled 'glucose present'. TABLE 2. The effect of glucose on rate of water absorption from the isolated intestine in the presence of different 'volume indicators'. Distension pressure, 45 cm H20. Other experimental conditions as in Table 1 Volume indicator Water absorption (1l./cm/hr) A Conc. Osmotic Glucose Glucose (mm) effect (cm H20) present absent Urea 20 56 213±42 (6) 26±36 (6) Creatine 0*4 3 178±36 (4) 6±22 (3) Sorbitol 55 810 190±18 (4) 20±22 (4) The effect of glucose on water absorption at 45 cm H20 distension pressure was also measured using creatine and sorbitol as volume indicators. These measurements, together with the corresponding ones from Table 1, are shown in Table 2. This table also gives the approximate concentrations in which the volume indicators were present in the inner fluid, and their 'osmotic effects' which depend on their concentrations and on the extent to which they are retained in the lumen. These osmotic effects are referred to more fully in the discussion. The results with the different volume indicators are sufficiently similar to indicate that the effect of glucose on water absorption by this particular intestinal preparation is not dependent on any peculiar properties of the foreign substances introduced as volume indicators. The results also confirm that the rate of water absorption is extremely small in the absence of glucose. Absorption of urea, creatine and sorbitol The data used to estimate water absorption also give estimates of rate of absorption of the volume indicator. These estimates are best presented by expressing the amount of volume indicator absorbed as a percentage of the 42 PHYSIO. CXXX 'A-

658 R. B. FISHER amount present in the inner fluid at the beginning of the experiment and relating this to the percentage of the original volume of water which is absorbed in the same time. The results are shown in Table 3. It is seen that the presence of glucose in the inner fluid encourages the absorption of solute in parallel with that of water. Except perhaps in the instance of urea, there is negligible absorption of solute in the absence of glucose. It is possible that the urea results are influenced by partial decomposition of urea by remnants of food urease, and that some of the urea is absorbed as ammonia. TABLE 3. The relation between the rate of absorption of water and that of urea, creatine and sorbitol in the presence and absence of glucose in the inner fluid. Data from the experimiients of Table 2. Percentages of water and solute absorbed Glucose present Glucose absent Water Solute Water Solute Solute (W) (S) 100 (S/W) (W) (S) Urea 20-1 18-0 90+10 (8) 2-4 6-5 Creatine 21-7 14-6 67± 3 (4) - 1-6 0-2 Sorbitol 29-3 10.9 36± 6 (4) 4-1 -7 The ratio IOOS/W, where S is percentage of solute absorbed and W is the percentage water absorbed, gives the concentration of the solute solution absorbed as a percentage of the concentration of the solution in the intestinal lumen. The magnitude of this ratio falls with increasing molecular weight of the solute. A few observations have been made at concentrations other than those used ordinarily (i.e. those shown in Table 2). Two experiments with 11 mm-sorbitol, instead of 55 mm, gave a mean value of loos/w of 36, identical with that found for the higher concentration, and four experiments with 5 mm-urea instead of 20 mm gave a value of 100 + 18 compared with 90 + 10. The osmotic changes during water absorption Although the change in distension pressure which has been studied is without significant effect on water absorption, the experiments cited do not rule out the possibility that water absorption may be the passive resultant of combined osmotic and hydrostatic pressure differences across the mucosal epithelium. Since 100 mg/100 ml. of glucose exerts an osmotic pressure of approximately 125 cm H20, and since the final glucose concentration difference between inner and outer fluids may be over 200 mg/100 ml. at the end of an hour (Fisher & Parsons, 1950, and Table 5 of this paper) it is possible that osmotic pressure differences could be set up by the process of active glucose absorption which were much larger than the practicable range of hydrostatic pressure differences.

WATER ABSORPTION 659 Experiments have therefore been made in which freezing-point depressions have been measured on inner and outer fluids at the beginning and end of 60 min absorption periods. Some of these experiments have been made with 500 mg/100 ml. of glucose in both inner and outer fluids initially, and some with no glucose initially in the outer fluid. The results are given in Table 4. These experiments were made on female rats. There is no significant difference in osmotic pressure between inner and outer fluids at either end of the period of absorption when both fluids initially contain glucose, but when there is no glucose initially in the outer fluid the initial osmotic pressure difference (which is strongly adverse to water transfer out of the intestine) is diminished somewhat in size but not altered in sense by the end of the hour. The water and glucose absorption rates are very similar in the two sets of experiments. TABLE 4. Freezing-point depressions during absorption from inner fluid containing 500 mg/100 ml. glucose. Four experiments in each series: adult female rats Freezing-point depressions (thousandths of a degree) Mean rates of absorption Glucose conc., A_A Initially After 60 min in outer fluid Water Glucose, - A (mg/100 ml.) (dl./cm/hr) (mg/cm/hr) Inner Outer Diff. Inner Outer Diff. 500 229±23 2*52±0-13 626 623 3±5 619 618 1±4 nil 213±12 2-32±0-13 606 551 55±8 599 563 36±7 TABLE 5. The changes in glucose concentrations in the experiments of Table 4, together with their osmotic equivalents and comparison of the latter with osmotic pressure changes calculated from the freezing-point depressions Osmotic pressure differences: Mean glucose concentrations (inner - outer): cm H20 (mg/100 ml.) A from glucose from freezing- Glucose initially (Inner - concentration point in outer fluid Inner Outer outer) differences measurements Present Initial 465 488-23 -30 44 60 min 257 536-279 -359 9 Change - 329-35 Absent Initial 480 480 617 694 60 min 241 200 41 53 447 Change - - 564-247 The osmotic pressure differences of Table 4 are given in the last column of Table 5 in terms of cm H20. These figures make it apparent that water absorption can continue unaffected by an adverse osmotic pressure gradient which is enormously greater than any favourable hydrostatic pressure gradient which could be set up across the mucosal epithelium in any circumstances. The observed osmotic pressure changes are not solely dependent on changes in glucose concentration. Table 5 gives the observed glucose concentrations at the beginning and end of the period of absorption, and the osmotic equivalents of the concentration differences. It is seen that the movement of glucose out of the intestine might be expected to diminish the osmotic force holding water 42-2

660 R. B. FISHER in the lumen by 329 and 564 cm H20 respectively in the two series of experiments, with and without outer fluid glucose, whereas the observed diminutions are only 35 and 247 cm H20 respectively. Thus the observed changes are 294 and 317 cm H20 less than expected on the basis of glucose movement, indicating that other active osmotic changes are taking place to more or less the same extent in the two series of experiments. The relation between glucose absorption and water absorption Since the observed dependence of water absorption on glucose absorption might signify that the water absorption was an integral part of the process of glucose absorption, experiments have been made in which the glucose concentration in the lumen was reduced to 100 mg/100 ml., and additions of known volumes of medium containing glucose in this concentration were made at 15 min intervals. In this way it proved possible to reduce the mean rate of glucose absorption over hour periods to 1*08 + 0-09 mg/cm/hr in six experiments, whilst maintaining glucose absorption throughout the period. The mean rate of water absorption was 254 + 26,u1./cm/hr. These experiments were made on female rats, so that these figures are to be compared with those of Table 4. This procedure reduces glucose absorption to about 40 % of the figure in Table 4, but the water absorption rate is not diminished. An attempt was made to supply the metabolic requirements of the mucosa by adding 2 g/100 ml. of glucose to the outer fluid, omitting glucose from the inner fluid. In these experiments there was no measurable water absorption. There was enough glucose to cause a 2500 cm H20 osmotic pressure difference between inner and outer fluids, in favour of water transfer. Smyth & Taylor (1955) have recently shown that very small concentrations of phlorizhin inhibit water absorption from surviving intestine. Experiments were made to determine whether there was any dissociation between the effect of phlorizhin on water absorption and its effect on glucose absorption. These experiments were made on male rats by introducing an exact volume into the inner circuit and measuring the volume and glucose content of the fluid drained out after an hour. The results are given in Table 6. It is obvious that there is a close parallelism between glucose and water absorption rates. TABLE 6. Effect of phlorizhin on absorption from the intestine of glucose and water. Experiments on female rats. Phlorizhin Glucose Water concentration absorption absorption (mg/100 ml.) (mg/cm/hr) (Il./cm/hr) 0 2-52 194 0 2-34 177 0 2*25 175 3 1-12 66 3 0 93 52 10 0-62 40 10 0-52 15

WATER ABSORPTION 661 DISCUSSION These experiments show that simple diffusion and filtration do not account for the absorption of water, urea, creatine and sorbitol from the lumen of the surviving small intestine of the rat. The absorption of water is an active process, continuing unabated against an adverse osmotic pressure difference of 400-700 cm H20, and this active process, though dependent on glucose absorption, is not at all closely dependent on its rate. The nature of the effect of glucose on water absorption It has been shown (Fisher & Parsons, 1953) that the relation between glucose concentration in the lumen and the rate of glucose disappearance strongly suggests that a single process is responsible, although the intestine treats glucose presented to the lumen surface of the mucosa in two different ways: it transfers some glucose from inner to outer fluid and it metabolizes some. The single process resulting in loss of glucose from the lumen must be that of transference, and it has to be supposed that the majority of the epithelial cells, not being engaged in this transference, are not readily permeable to glucose at their free borders. A consequence of this is that supply of glucose to the basal ends of these cells (which are the portals of entry of nutrients in the intact animal) will be far more plentiful in the isolated intestine preparation when glucose is being absorbed from the lumen, and is being actively injected into the tissue spaces, than when it must diffuse across the whole thickness of the intestinal tissues from the outer fluid in order to supply the mucosa. This explanation is supported by the experiments with reduced glucose concentration in the lumen. Any more immediate relation between glucose absorption and water absorption would be difficult to reconcile with the observation that the rate of glucose absorption can be cut to 40 % without affecting the rate of water absorption. The failure to support glucose absorption by supply of glucose in high concentration from the outer fluid is not a serious bar to acceptance of this conclusion. It need not mean more than that the difficulties of diffusion across the submucosal tissues are too great to permit of adequate supply by this route. The phlorizhin experiments are also inconclusive. If this substance inhibits the catabolism of glucose, and so limits the energy supply of the mucosal epithelial cells, it might be expected to inhibit all cellular secretory activities. Only if water absorption and glucose absorption happened to be mediated by different types of cell with very different susceptibilities to -phlorizhin could these experiments have yielded useful evidence.

662 R. B. FISHER The relation of water absorption to osmotic pressure differences The evidence presented in this paper that water absorption is not reduced by large osmotic pressure differences or increased by large favourable ones does not mean that osmotic effects on water absorption are not possible, but merely that they are only produced by very large deviations from normal osmotic relations. G. J. R. McHardy & D. S. Parsons (personal communication) have shown that, when fluid perfused through the lumen of the rat intestine in situ contains half-isotonic mannitol, water absorption is almost completely inhibited. This concentration of mannitol will exert an osmotic pressure of about 3600 cm H20, and if mannitol behaves like sorbitol it will have an 'osmotic effect' equivalent to about two-thirds of this, i.e. about 2400 cm H20. The osmotic effect is computed on the basis that since the concentration of sorbitol in the solution absorbed is about one-third the concentration in the lumen, the sorbitol behaves as if two-thirds of it were incapable of leaving the lumen. Reference to Table 2 shows that the osmotic effect of mannitol in McHardy & Parsons's experiments is probably three times as great as the highest in any of the present experiments. The experiments made with no glucose in the inner fluid and 2 % glucose in the outer fluid, in which the glucose is exerting an osmotic pressure of 2500 cm H20 in the direction favourable to water absorption, showed no effect of the glucose on water absorption. It seems likely then that the effects of high osmotic pressure are indirect. They can slow water absorption by increasing the work required to translocate unit mass of water, but they cannot increase water absorption by exerting an osmotic pull. Presumably the transfering cells are already working nearly maximally. The absorption of solute molecules The absence of absorption of urea, creatine and sorbitol in the absence of water absorption indicates that no specific mechanisms exist for the absorption of these substances, and also suggests that diffusion across the mucosal epithelium must occur with difficulty-a suggestion in accord with the demonstrated lack of dependence of water absorption on hydrostatic and osmotic factors. The absorption of these solutes, when water is being absorbed, at rates proportional to their concentrations in the lumen suggests that they are somehow entrained in the stream of absorbed water. Any active water absorption mechanismwhich depended on actively reducing the water concentration inside the cell at that pole at which water is taken up would necessarily and non-specifically raise the concentration of intracellular solutes in the same region. It would be expected that a diffusion gradient of solutes would be set up in the direction opposite to that in which the water

WATER ABSORPTION 663 tended to move. It is difficult to see how such a mechanism could cause solutes to move in the same direction as water. No satisfactory solution to this difficulty exists, but two suggestions can be made. If it were possible to show that the canaliculi believed to occur in the brush borders of the mucosal epithelial cells possessed mechanical pumping powers, then it might be possible to envisage them as pumping lumen fluid through molecular sieves into the cells. Provided that the pumps were intermittent, such a mechanism would account for the apparently non-specific entrainment of small molecules in the water stream and for the fall in relative concentration of the solute in the fluid absorbed as the molecular weight of the solute rises. The other possible mechanism is that suggested by Goldacre (1952), inter alia, namely that a reversible denaturation of protein occurs in the absorbing cells, at the pole at which water and solutes enter, and that this denatured protein has a far greater capacity to combine with water and other molecules than the native protein. If the volume changes consequent on denaturation set up protoplasmic streaming the denatured protein will be carried to the other pole of the cell where renaturation is conceived to take place with release of the water and solutes transported. The establishment of this hypothesis would require the demonstration of protoplasmic streaming in mucosal epithelium cells, and one would also expect that the relative rates at which different solutes were transported would be more dependent on chemical peculiarities than on molecular size. This last point is not a strong objection, since the present data are inadequate to establish the dependence on molecular size at all firmly. Whatever the basis for the coupled absorption of water and small solute molecules, it has an interest of its own. In view of the difficulty with which diffusion out of the intestinal lumen takes place, some such process as this coupled absorption must be the basis of the absorption of the majority of drugs and of many small molecules of natural origin to be found in foodstuffs, and it may be supposed that physiological and pathological factors which affect water absorption will affect equally the absorption of this large class of solutes. Sex differences in intestinal absorption A curious by-product of this work is the observation that the rate of absorption of water and of glucose per cm length is markedly greater in female than in male rats. The mean water absorption rate of fourteen female rats was 236,u./cm/hr and that of eighteen males was 188. The mean glucose absorption of eight females was 2-42 mg/cm/hr and Fisher & Parsons (1950, 1953) have previously reported figures for males of 18-1-9. The difference is not due to difference in intestinal dimensions: in a large series of observations it has been found that the mean length of the whole small intestine in the rat is about 10 %

664 R. B. FISHER longer in the female than in the male. Thus the absolute capacity of the small intestine to absorb water and glucose is about 40 % greater in the female than in the male. This does not mean that rates of absorption in vivo will be in these proportions. These figures refer to conditions in which the whole of the mucosa is constantly exposed to the solution to be absorbed, and this cannot happen in vivo. SUMMARY 1. No absorption of water occurs from the surviving small intestine of the rat even under 45 cm H20 distension pressure unless the fluid in the lumen contains glucose. 2. Urea, creatine and sorbitol are not absorbed unless water is being absorbed at the same time. When they are absorbed, the solution leaving the lumen is more dilute than that in the lumen, the difference in concentration increasing with increase in molecular weight. 3. Changes in osmotic pressure set up by active glucose absorption do not account for the effect of glucose on water absorption. 4. The rate of glucose absorption can be reduced substantially without reducing water absorption. 5. It is concluded that water absorption is wholly an active process, and that the role of glucose is largely or entirely nutritive. REFERENCES ENNOR, A. H. & STOCKEN, L. A. (1948). The estimation of creatine. Biochem. J. 42, 557-563. FsHER, R. B. (1954). The absorption of water and of some non-electrolytes from the surviving small intestine of the rat. J. Physiol. 124, 21-22P. FISHER, R. B. & LINDSAY, D. B. (1955). The action of insulin on the penetration of sugars into the perfused heart. J. Physiol. (In the Press.) FISHER, R. B. & PAsoNs, D. S. (1949). A preparation of surviving rat small intestine for the study of absorption. J. Phy8iol. 110, 36-46. FismHE, R. B. & PA.ONS, D. S. (1950). Glucose absorption from surviving rat small intestine. J. Phy8iol. 110, 281-293. FISHER, R. B. & PARSONS, D. S. (1953). Glucose movements across the wall of the rat small intestine. J. Phy8iol. 119, 210-223. GOLDACRE, R. J. (1952). The folding and unfolding of protein molecules as a basis for osmotic work. Int. Rev. Cytol. 1, 135-164. SMYTH, D. H. & TAYLOR, C. B. (1955). The inhibition of water transport in the in vitro intestinal preparation. J. Phy8iol. 128, 81-82P. WELLs, H. S. (1931). The passage of materials through the intestinal wall. I. The relation between intra-intestinal pressure and the absorption of water. Amer. J. Physiol. 99, 209-220.