from the gastro-intestinal tract. Benson et al. [1956] have suggested that

Transcription

1 Quart. J. exp. Phy8iol. (1967) 52, 19-3 THE ROLE OF THE LYMPHATIC SYSTEM IN THE ABSORPTION OF WATER FROM THE INTESTINE OF THE RAT. By J. BARROWMAN and K. B. ROBERTS. From the Department of Physiology, The London Hospital Medical College, London, E.1. (Received for publication 1th May 1966) The flow and composition of lymph in the cisterna chyli and mesenteric lymphatic vessel of the restrained conscious rat has been studied in relation to the ingestion of 5 ml. of water and isotonic saline. In both cases the lymph flow increases and the protein concentration of the lymph falls; the time relationships are, however, different. When water is taken, the response begins in a few minutes and is over in 3 minutes; when saline is taken, the response begins in 15 minutes and is over in 6 minutes. The excess amount of lymph recovered during the responses is about -8 ml. These studies, and studies with tritiated water, suggest that during the first 1 minutes after rats drink water there is a transient bulk flow of fluid from the intestinal lumen into the intestinal lymphatics. The intestinal lymphatics seem to act as an overflow system during the absorption of hypotonic fluid. THERE is no general agreement as to the relative importance of the portal venous and intestinal lymphatic route in the transport of absorbed water from the gastro-intestinal tract. Benson et al. [1956] have suggested that about 99 per cent of water is absorbed into portal venous blood and only 1 per cent or less into the mesenteric lymphatics. This conclusion was based on work in conscious rats. Nevertheless in vitro studies by Lee [1961, 1963, 1965] have indicated that intestinal lymphatics may play a more important role in water absorption. It has been known for some time that water ingestion is associated with an increased flow of thoracic duct lymph; this has been studied in particular by Simmonds [1954] in the rat, though it has also been noted in other species such as the dog [Watkins and Fulton, 1938] and man [Crandall et al., 1943]. In the present studies, conscious rats with chronic lymphatic fistulae, either of the cisterna chyli or a mesenteric lymph vessel, were used in an attempt to resolve these conflicting ideas. Preliminary reports of this work have already appeared [Barrowman and Roberts, 1965 and 1966]. METHODS Preparation of chronic lymphatic fistulce (a) Cisterna chytifistulce. - Adult laboratory rats, of the Sprague-Dawley or Wistar strains, weighing approximately 25 g. were used. Under ether anaesthesia a fine polyethylene cannula was placed in the cisterna chyli pointing caudally [Bollman et at., 1948]. At the same time, an intravenous infusion into the left femoral vein of 9 per cent NaCl solution was set up. A soft plastic tube was used for this (Esco Rubber Co., Ltd.). When the rat had recovered from the anaesthetic, it was placed in a restraining cage of the type described by Boilman [1948], and for the remainder 19

2 2 Barrowman and Roberts of the day was kept warm and shielded from the light. The intravenous infusion was continued until the following morning, each animal receiving a total of between 5 and 1 ml. of intravenous fluid. (b) Mesenteric lymphatic fistulce. - The largest of the mesenteric lymphatic vessels was exposed in the way described by Bollman et al. [1948] and cannulated with fine polyethylene tubing. This vessel drains the small, and perhaps a portion of the large, intestine but not the stomach or other viscera [Job, 1915]. The tubing was passed through a stab wound in the right flank of the animal, no attempt being made to fix it. In other respects, these animals were treated in the same way as those with cisterna chyli fistulae. Experiments on these animals with chronic lymphatic fistulae were carried out between the second and fifth post-operative days. Lymph flow measurements. - A drop-recorder was used and a permanent record obtained on a kymograph. Large volumes of lymph were measured directly in a volumetric cylinder, whilst specimens for chemical analysis were weighed in stoppered bottles and their volumes calculated using a specific gravity of 1 6, a mean figure derived from measurements made on a series of assorted lymph specimens. Variations of specific gravity observed during the experiment described do not alter to any significant extent the volumes calculated from the weights of specimens. Arterial blood specimens. - A polyethylene cannula was introduced into the aorta via a stab wound made with a needle. The tip of the cannula was passed caudally. Small amounts of heparin solution prevented clotting in the cannula; the conscious animals were restrained in the same way as those with lymphatic fistulae. Samples of aortic blood were taken, 2 days after the operation, during the course of water absorption. Lymph turbidity. - Measurements of the optical density of diluted specimens of lymph were made at a wavelength of either 3 or 34 m,t in a 'Unicam' SP 5 spectrophotometer. Protein estimations. - The method employed was a modification of the biuret test of Weichselbaum [1946], as adapted for spinal fluid protein estimation by Dittebrandt [1948]. This requires optically clear solutions. Five-drop specimens of lymph were weighed, diluted with 3 ml. of 9 per cent NaCl and then centrifuged at 15, g. for 25 minutes in cellulose nitrate tubes. The fat aggregated as a supernatant and the tubes were cut just below this layer with a sharp knife blade which also sealed off the two sections of the tube. The lymphocytes in the lymph formed a very small pellet at the bottom of the tube. Glucose estimations. - These were performed on 5 or -1 ml. samples of lymph by the method of Nelson [1944] and Somogyi [1945]. Sodium estimations. - Specimens of lymph diluted with glass distilled water were used. The concentration of sodium was estimated at 589 m, in a Zeiss flame photometer, PMQ II - MQ4 III. Tritiated water experiments. - Animals drank water containing approximately 18, curies of tritium per ml. Weighed, five-drop, specimens of blood or lymph were collected in 1 per cent trichloracetic acid. After centrifugation, the protein-free supernatant was added to a mixed solvent phosphor [Kinard, 1957] and the amount of tritium estimated in a liquid scintillation counter (Nuclear Enterprises Ltd., 834). In presenting these results, we have taken the concentration of tritium in water as 1 per cent and have expressed the activity of the lymph and blood specimens as a fraction of this. In all cases where chemical estimations are plotted together with flow rates of lymph, allowance has been made for the dead-space of the cannula. Each point on the graphs, therefore, represernts the concentration of the substance in question in the lymphatic vessel at the mid-point of the period over which the specimen of lymph was collected.

3 Lymphatics and Water Absorption 21. RESULTS An increased flow of cisterna chyli lymph occurs when restrained rats drink. This response was investigated by standardizing the amounts of fluid given. After a period of from 6 to a maximum of 2 hours of fluid deprivation, rats would drink readily. Moreover, after such fluid deprivation, the flow of lymph was slow and constant in an individual animal, usually amounting to SPEC TROPHOTOMETER READING AT 34mp \.4 _. 16 DROPS/5 MIN so tt. TIME (MIN.) S mlwat ER ORA LLY FIG. 1. Cannulla idthe cisterna chyli. Rat given 5 ml.of water. Above: the optical density of successive 5-drop specimens of lymph. sample was diluted by the addition of 3 ml. of 9 per cent NaCl. Below: rate of flow of lymph. Each about -8 ml. per hour. It was found that these animals would take 5 ml. Of fluid in about 5 minutes, and this volume was chosen. When water was taken, the flow of lymph increased rapidly but the response was completed within half an hour (Table I). The pattern when.9 per cent NaCl was drunk was quite different; there was a lag period of 1 to 15 minutes followed by a more prolonged response. Similar patterns in each case were obtained from rats having mesenteric lymphatic fistulae. The results from both types of piteparation are summarized in Table I. The lymph that flows after the rats drink water or saline is, to the naked eye, less milky than that flowing before and this was confirmed by measuring the optical density of suc- driniking, cessive samples of lymph (fig. 1). Protein estimations. - Fig. 2 illustrates the changes in protein concentration and protein output of the lymph before, during and after the ingestion of

4 A t o ~~~~~~~~~m + _ 22 Barrowman and Roberts m~~~~~~~~~t.ob> C) oc Eq~~~~~~e O E- S + + C H - t_ P4 >b E- 4z C O Oo O x~~~~qx P4 A.@ } s x EHVee O

5 Lyniphatics and Water Absorption LYMPH PROTEIN OUTPUT mg./mim. 1.6 LYMPH PROTEIN OUTPUT mg/min i *. j'i li'l i o" 1.2.O i o-o I 7.e... /11, 1.1% ~ LYMPH PROTEIN CONCN. G./1ml. R..P a. 2 * DROPS/S MIN DROPS/5 MIN O IL I'll, I '' ", "i.,, o t t 5 m,t WAT ER ORALLY. t t 5 ml.9% NaCl ORALLY. T*IME ( MINS l FIG. 2. Cannula in the cisterna chyli. (Two different rats). Left: rat given 5 ml. of water. Protein output, protein concentration and rate of flow of lymph. Right: rat given 5 ml. of -9 per cenit NaCl. The same measurements.

6 24 Barrowman and Roberts water in a cisterna chyli preparation. It is to be noted that all the lymph flowing from the cannula was collected as successive 5 drop samples and LYMPH PROTEIN OUTPUT m9/min LYMPH PROTEIN OUTPUT mg/min I/.4 4 LYMPH PROTEIN CONCN. G/1 mt LYMPH PROTEIN CONCN. G/1ml 3 4 l 2~~~~~ * C 24- DROPS/S MIN. -U DROPS/SMIN ,r-> 7TV; 8..j/ >,.',,,, < 2 2 t SSml WATER ORALLY 4, I- "I*.,.1i. I,I,I i, t t.sml.9% NaCL ORALLY TIME MIN., FIG. 3. Cannula in a mesenxteric lymphatic vessel. (Two different rats). Left: rat givenx 5 ml. of water. Protein output, protein concentration and rate of flow of lymph. Right: rat given 5 ml. of -9 per cent NaCl. The same measurements. protein estimations were carried out on each sample. From this, and the rate of flow of lymph, the output of protein has been calculated. It can be seen that there is a dilution of lymph with respect to protein which parallels the increase in flow. There is an increased output of protein but this starts

7 Lymphatics and Water Absorption 25 to fall while the flow rates are still high. Similar results were obtained in 8 other experiments. Fig. 2 also shows the same measurements made in a rat drinking.9 per cent NaCl. The fall in protein concentrations once again parallels changes in flow. In this case, the lymph protein output remains LYMPH CONCN. GLUCOSE mg/1ooml 81 4! 24- DROPS/S MIN a o..,. D...X,d ml t t TTIME M IN.' WATER ORALLY FIG. 4. Cannula in a mesenteric lymphatic vessel. Rat given 5 ml. of water. Above: the concentration of glucose in successive samples of lymph. Below: rate of flow of lymph. high throughout the response. Similar results were obtained in 4 other experiments. Fig. 3 illustrates the changes in protein concentration and outputs in a mesenteric lymphatic preparation, and it can be seen that the results are comparable with those using cisterna chyli preparations. Similar results were obtained in 6 other experiments. Glucose and sodium estimations. - The enhanced flow of intestinal lymph following the ingestions of water was, as has been shown, associated with a fall in turbidity and with a dilution of lymph protein; no such dilution was observed, however, with respect to either glucose (8 experiments) or the sodium ion (4 experiments).

8 26 Barrowman and Roberts Figs. 4 and 5 illustrate representative experiments of this type. Tritiated water estimation8. - Following the ingestion of 3H2O, the isotope appears rapidly in the lymph. In 6 experiments using cisterna chyli preparations and in 5 using mesenteric lymphatic preparations, the concentration of tritium in the lymph rose to a peak of between 2 and 6 per cent within the first 1 minutes and returned to a plateau of between 1 and 3 per cent within LYMPH Na 4 CONCN. mg/looml DROPS/5 MI N. B o 2 2 TIME MIN.) Smi WATER ORALLY FIG. 5. Cannula in the cisterna chyli. Rat given 5 ml. of water. Above: the concentration of sodium in successive samples of lymph. Below: rate of flow of lymph. 2 minutes. Examples from both types of preparations are shown in fig. 6. In two experiments following the ingestion of.9 per cent NaCl made up in tritiated water, there was a slower rise in the isotope in the lymph, a plateau being reached - without a preceding peak - at approximately 2 minutes (fig. 7). DIscuSSION The experiments reported in this paper are of the changes in the flow and composition of lymph leaving the intestine when conscious rats voluntarily drink water or saline. These rats were deprived of fluids for some hours prior to the experiment. Experiments using in vitro preparations, or experiments on anesthetized rats, or experiments in which fluids are injected under pressure into the gut of well or over-hydrated rats, are not necessarily comparable.

9 Lymphatics and Water Absorption 27 A consistent and striking change seen in our experiments is the rapid flow of dilute lymph in the cisterna chyli following the ingestion of water. A similar phenomenon was described by Simmonds [1954] where water was injected through a gastrostomy. In our experiments the extra volume of lymph in excess of basal amounts was, as a mean,.8 ml. after 5 ml. of water %oj LYMPH AS 3H O LYMPH AS 3H2 2 ~~~~~~~~~~ DROPS/ 4 5 MIN 32 / ~~~~ DROPS/5MINO I/ 4 lh/ral were~~ taen Insm nml,hwvr h eoeywsa uha. ml.an Mi thr H2 ORlY aralmunsrcrddmyb M- TIM IN.) elated Cato ihestate l ofihydration Aofvte -adioactiit trtargerdrsalrthn5m.o waterwrgiven; Beothetflwoflmh rpercetaghee,voflumesi In otherlmp experientsedasot time relations of the responses to these were found to be similar to those in Table I. It would be interestinlg to relate the extra volumes of lymph recovered in such exrperiments to the amounts ingested in rats with carefully controlled hydration. This has yet to be done; it would require a relatively large number of animals.

10 28 Barrowman and Roberts The increase in the flow of lymph in the cisterna chyli could be the result of water passing from the intestinal lumen through the mucosa into the lymphatics of the gut. There are, however, other possibilities: the excess of fluid in the cisterna chyli might result from enhanced filtration of fluid from the blood to regions drained by the cisterna chyli. In particular, fluid might be absorbed into the hepatic portal vein from the intestinal lumen, pass to the liver and there be filtered into the hepatic system and thence into /,.of LYMPH 4 AS 3H O 3 2 I6 DROPS/5 MIN eC3z 2 /. 1A t t 3.5ml.97. NaCI IN H2 ORALLY TIME (MIN.) FIG. 7. Cannula in the cisterna chyli. Rat given 3-5 ml. of tritiated saline. Above: radioactivity in the lymph expressed as a percentage of that in the admini. stered solution. Below: rate of flow of lymph. the cisterna chyli. The experiments on the mesenteric lymphatic preparation eliminate the latter possibility since the vessel used drains effectively only the small intestine. The excess lymph can therefore come either from the gut lumen or the intestinal blood. In the latter case it could be the result of changes in haemodynamics, vascular permeability or a combination of these two. On many occasions, the increased flow of lymph following water ingestion occurred in about one minute after the animal began to drink. The earliest samples of lymph were diluted with respect to protein (see figs. 2 and 3). Such rapid dilution would be more readily explained in terms of bulk flow of water from the hypotonic lumen via the intestinal mucosa into neighbouring lymph vessels. This explanation would be quite consistent with the physiological function of the lymphatics as an overflow for interstitial fluid. The hypotonic fluid in the gut lumen will gradually become isotonic

11 Lymphatics and Water Absorption 29 [Follansbee, 1945] and this may account for the flow of lymph returning within 3 minutes to basal conditions. The concentrations of sodium and glucose do not show the dilution seen for protein. This may be due to the rapid exchange of these smaller solutes with the extracellular fluid. A rapid equilibration occurs also in experiments using water containing 3H2, for within 1 to 2 minutes the isotope drunk has reached a stable level in blood and lymph of 1 to 3 per cent, a value to be expected on the basis of even distribution through the entire body water. However, if the amounts of the radioactivity in the lymph are carefully followed for the first few minutes after the start of drinking tritiated water, a brief peak of isotope appears (fig. 6). This is also consistent with an initial and transient bulk flow of fluid from lumen to lymphatics. It is to be noted that the pattern of absorption of the isotope from.9 per cent NaCl made up in tritiated water is quite different, although there are presumably equal opportunities for diffusion. In this case (fig. 7), there is no peak preceding the plateau of even distribution. In a further series of experiments, aortic blood levels were taken in restrained animals after the ingestion of tritiated water. The amount of radioactivity appearing in the aortic blood followed a smooth curve rising to a plateau within 2 minutes, no peak being seen [Barrowman and Roberts, unpublished observations]. It is argued that when water is drunk by these rats, some of it passes rapidly into intestinal lymphatics without any mobilization of protein. The brief rise in protein output seen in figs. 2 and 3 following water ingestion may represent the washing out of protein-rich lymph present in the intestinal lymphatics by the tide of incoming fluid. The patterns seen when.9 per cent NaCl is drunk are quite different: the response is slower and excess protein is mobilized, a finding consistent with previous work [Simmonds, 1954]. In this case the increased flow of lymph might have a large component of excess fluid derived from the bloodstream. Reference has been made already to the work of Benson et al. [1956] which at first sight seemed to suggest that less than 1 per cent of water is absorbed by the lymphatics. However, this conclusion is derived from a different experimental procedure in which very small volumes (.2 ml.) of isotopic water were administered by gastrostomy to conscious rats. In some cases, the isotope was washed into the gastro-intestinal tract with isotonic fluid. Such experiments are more comparable with the present work in which 3H2O was given in saline. The samne considerations apply to the experiments of Noyan [1964] who observed the uptake of labelled water from an isotonic solution. Lee [1953] found that lavage of the small intestine of the dog with water greatly increases the lymph flow from that segment compared with lavage with isotonic saline. If the intestinal lymphatics act as an overflow system, as we suggest, the proportion of absorbed water passing by this route will depend on the amount and tonicity of the fluids presented to the gut mnucosa and the state of hydration; it will not be a fixed proportion of the amount drunk and attempts to determine a fixed proportion would be misleading.

12 3 Barrowman and Roberts ACKNOWLEDGMENTS We are grateful to Miss Susan Young for skilled technical assistance. Miss D. Goodman estimated the sodium. We are indebted also to Professor J. L. Gowans, F.R.S., Professor C. J.. R. Morris and Dr. J. Fawcett for advice and use of some apparatus. Professor K. W. Cross and Dr. P. Goodford kindly read the manuscript. REFERENCES BARROWMAN, J. and ROBERTS, K. B. (1965). J. Physiol. 177, 12-13P. BARRoWMAN, J. and ROBERTS, K. B. (1966). J. Physiol. 182, 33-34P. BENSON, J. A. (Jr.), LEE, P. R., SCHOLER, J. F., KIM, K. S. and BOLLMAN, J. L. (1956). Am. J. Physiol. 184, BoLLmAN, J. L. (1948). J. Lab. clin. Med. 33, Bo.LMAN, J. L., CAIN, J. C., and GRINDLAY, J. H. (1948). J. Lab. clin. Med. 33, CRANDALL, L. A. (Jr.), BARKER, S. B. and GRAHAM, D. G. (1943). Gastroenterology 1, DITTEBRANDT, M. (1948). Am. J. clin. Path. 18, FOLLANSBEE, R. (1945). Am. J. Physiol. 144, JOB, T. T. (1915). Anat. Rec. 9, KINARD, F. E. (1957). Rev. scient. Instrum. 28, LEE, J. S. (1953). Ph.D. Thesis, University of Minnesota, cited by M. B. Visscher in 'Metabolic Aspects of Transport across Cell Membranes' edited by Q. R. Murphy. The University of Winsconsin Press, Madison LEE, J. S. (1961). Am. J. Physiol. 2, LEE, J. S. (1963). Am. J. Physiol. 24, LEE, J. S. (1965). Am. J. Physiot. 28, NELSON, N. (1944). J. biol. Chem. 153, NOYAN, A. (1964). Proc. Soc. exp. Biol. Med. 117, SimnmoNDs, W. J. (1954). AuSt. J. exp. Biol. med. Sci. 32, SoMoGYI, M. (1945). J. biol. Chem. 16, WATKINS, A. L. and FULTON, M. N. (1938). Am. J. Physiol. 122, WEICHSELBAIuM, T. E. (1946). Am. J. clin. Path. (Technical Section) 1, 4-49.