found it difficult to express all the fluid from the loop. 32-2

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1 487 J. Physiol. (I940) 98, I 6i2.364: THE ABSORPTION OF WATER FROM THE COLON OF THE RAT UNDER URETHANE ANAESTHESIA By B. L. ANDREW, J. N. DAVIDSON AND R. C. GARRY From the Physiology Department, University College, Dundee, The University of St Andrews (Received 20 June 1940) THE colon of the rat under urethane anaesthesia can absorb only negligible amounts of monosaccharides [Davidson & Garry, 1939]. In view of the rather unexpected nature of this result, we decided to investigate the powers of the colon to absorb other substances, in particular water. Moreover, we wished to make sure that the colonic loops in such rats had still an active circulation and that absorption was therefore possible. Three methods of attack were chosen. In the first place, since sugars are not absorbed from the colon, the variation of concentration of sugar in a closed loop of colon will give an indication of the movement of water into or out of the loop. Secondly, a non-absorbable indicator will reveal increase or decrease in the concentration of the contents of the loop in similar fashion. The indicator used was phenol red first recommended by Goreham in 1923, and subsequently used by a large number of investigators, e.g. Wilhelmj, Neigus & Hill [1933], Penner, Hollander & Saltzman [1938], as a dilution indicator in gastric analysis. The concentration of phenol red employed was too low to have any appreciable effect on the osmotic pressure of the solution. Thirdly, deuterium oxide may be used as an index of water exchange as has been done in the case of the small intestine by Hevesy & Hofer [1934], McDougall, Verzar, Erlenmeyer & Gaertner [1934] and by Peters & Visscher [1939]. Distilled water alone cannot be used since it damages the mucous membrane [Dennis, 1940]. We therefore introduced as solute either glucose or xylose in all experiments. No attempt was made to&measure the actual volume of solution in the colon at the end of 90 min., since we found it difficult to express all the fluid from the loop. 32-2

2 488 B. L. ANDREW, J. N. DAVIDSON AND R. C. GARRY METHODS Rats were given water but no food for 48 hr. and were then anaesthetized with urethane given subcutaneously in a dose of 1-6 mg./g. body weight. One hour later the abdomen was opened by a mid-line incision and the lower end of the ileum was tied off. An opening was made into the tip of the caecum and into the colon at its junction with the caecum. Colon and caecum were then washed out with Ringer's solution at 380 C. Thereafter a purse-string suture was inserted round the anus and tightened on a small glass cannula. A second cannula was inserted into the proximal end of the colon and all traces of Ringer's solution washed out of the colon with a stream of fluid containing hyper-, iso- or hypotonic sugar solution at 380 C. In the majority of experiments this sugar solution contained phenol red. The anal cannula was then withdrawn and the purse-string suture tightened. The colon was allowed to fill with solution, the upper cannula was withdrawn and the opening ligatured. In many cases the caecum was similarly washed out and filled with sugar solution. The abdomen was closed and the animal left in warm surroundings for 90 min. At the end of that tima the rat was killed, the colonic loop and the caecum were removed, opened and the contents drained out. Reducing sugar was estimated by the method of Hagedorn and Jensen and phenol red by the colorimetric method of Hollander, Penner & Saltzman [1937]. In the experiments with deuterium oxide the colon alone was used. After the colon had been washed out with Ringer the bulk of the residual liquid was removed with a few puffs of air. 5 ml. of a 4% solution of xylose in water containing approximately 20% D20 were then run repeatedly through the colon. The anal suture was tightened and about 3 ml. of the solution left in the colon which was then tied off as before. The remaining 2 ml. were preserved for control estimations of sugar and D20. At the end of a given time the animal was killed, the colonic loop removed, and the contents drained out. For the deuterium oxide estimations portions of about 1 ml. of the fluid under investigation were distilled in vacuo at 200 C. into a receiver cooled in a freezing mixture. When all the water had passed over, the distillate was allowed to come to room temperature and the D20 content was measured in a Pulfrich refractometer. Control experiments were carried out to ensure that no interfering substances were carried over in the distillation of fluid from colonic loops.

3 ABSORPTION OF WATER FROM THE COLON 489 RESULTS In all cases where the original solution was hypertonic (10.0%) the contents of the colon at the end of 90 min. were much less concentrated. Since passage of sugar out of the colon does not occur there must have been passage of water into the loop. When phenol red was present as well as glucose, the extent to which this indicator was diluted corresponded closely to the degree of dilution of the glucose (Table I). The opposite effect was seen where the initial glucose solution was hypotonic (2-7 %), the solution undergoing concentration during the 90 min. in the colon. The concentration of phenol red rose correspondingly (Table I). TABLE I. Variations in the concentration of the contents of colonic loops in rats under urethane anaesthesia in the course of 90 min. The figures given are average values obtained from several experiments. Glucose concentration Phenol red concentration g./100 ml. mg./100 ml. Initial Final Initial Final C, c2 c2/cl c, ' C2 c27/61 / * * *42 Where the initial glucose concentration was 5-4 %, the value usually considered to be isotonic with mammalian blood [Verzar & McDougall, 1936], the concentration within the colon after 90 min. was slightly lower, about 4-9 %. This was not due to loss of sugar since the phenol red concentration fell to a similar degree (Table I). Similar results were obtained with the caecum. In the experiments with heavy water, xylose was used as the nonabsorbable sugar. Xylose behaved in exactly the same way as glucose, and the strength of solution neither concentrated nor diluted was found to be 4 0 %. This, as in the case of glucose, is lower than the concentration (4.5%) said to be isotonic with blood. We therefore used a 40 % solution of xylose in water containing about 20-0% D20. The diminution in concentration of D20 due to exchange of deuterium atoms with the hydrogen atoms of the hydroxyl groups of xylose was negligible. Moreover any rapid exchange of deuterium atoms with hydrogen atoms present in the colon mucous membrane was allowed for by preliminary repeated passage of the solution through the loop. Although measurements of heavy water concentration by a refractometer are not particularly delicate, yet the 32-3

4 490 B. L. ANDREW, J. N. DAVIDSON AND R. C. GARRY concentration changes we observed were so gross that the method was perfectly adequate for the purpose. The xylose concentration, originally in the neighbourhood of 4-0%, remained at that level throughout the absorption period which varied from 5 to 90 min. We can therefore assume that the total amount of water in the colon remained constant. Deuterium oxide disappeared very rapidly from the colon; in 30 min. the bulk had gone, and even in as short a time as 5 min. a large proportion had disappeared (Fig. 1) Fig. 1. Min. Decrease in the concentration of heavy water in the contents of colonic loops in the anaesthetized rat. DISCUSSION There can be no doubt that the colonic mucous membrane in rats under the conditions of our experiments allows mass movement of water in both directions while remaining impermeable to monosaccharides. This latter property is lost when the colon is isolated in vitro [Davidson & Garry, 1939]. Even in the absence of an osmotic gradient, exchange of water molecules between the blood and the colonic contents was very rapid. The colonic mucous membrane seems to act as an extremely efficient semi-permeable membrane and there can no longer be any suspicion that the failure to absorb monosaccharides was due to experimental conditions interfering with the state of the colon.

5 ABSORPTION OF WATER FROM THE COLON 491 SUMMARY The colon of the rat under urethane anaesthesia, although impermeable to monosaccharides, is freely permeable in both directions to water molecules. We wish to express our thanks to Prof. W. F. K. Wynne-Jones for supplying the deuterium oxide and to the Carnegie Trust for the Universities of Scotland for an expenses grant for this work. REFERENCES Davidson, J. N. & Garry, R. C. [1939]. J. Phy8iol. 96, 172. Dennis, C. [1940]. Amer. J. Phy8iol. 129, 171. Goreham, F. D. [1923]. J. Amer. med. A88. 81, Hevesy, G. & Hofer, E. [1934]. Nature, Lond., 134, 879. Hollander, F., Penner, A. & Saltzman, M. [1937]. Proc. Soc. exp. Biol., N.Y., 36, 568. McDougall, E. J., Verzar, F. Erlenmeyer, H. & Gaertner, H. [1934]. Nature, Lond., i34, Penner, A., Hollander, F. & Saltzman, M. [1938]. Amer. J. dige. Diw. 5, 657. Peters, H. G. & Visscher, M. B. [1939]. J. ce. comp. Phy8iol. 13, 51. Verz4r, F. & McDougall, E. J. [1936]. Absorptionfrom the Intedtine. London: Longmans, Green and Co. Wilhelmj, C. M., Neigus, I. & Hill, F. C. [1933]. Amer. J. Physiol. 106, 381.