J. Physiol. (I953) 3121,

Transcription

1 452 J. Physiol. (I953) 3121, THE MOVEMENT OF POTASSIUM, SODIUM, CHLORIDE AND WATER ACROSS THE RUMEN EPITHELIUM OF SHEEP BY D. PARTHASARATHY AND A. T. PHILLIPSON From the Rowett Research Institute, Bucksburn, Aberdeenshire (Received 1 January 1953) Rumen liquor contains a high concentration of potassium (Parthasarathy, 1952 a), and in view of the known effects of this ion on cell membranes it was considered desirable to study the effect of solutions containing potassium in concentrations comparable to those of the rumen liquor, on the absorption of other ions and, in particular, of fatty acids. There is very little information to be found on the absorption of inorganic ions or of water from the rumen. Masson & Phillipson (1951) noted that when solutions containing no chloride were introduced into the rumen chloride appeared and they concluded it came from the blood. Scarisbrick & Ewer (1951) have reported on the irregular behaviour of inorganic phosphorus in the rumen, while recently Sperber & Hyden (1952), on the basis of experiments in which they studied the behaviour of solutions containing potassium, sodium, chloride and phosphate in a 'Pavlov' pouch of the rumen of goats, suggested that the rumen is selectively permeable to inorganic ions. The question whether water is absorbed from the rumen has never received close attention and, as there are good reasons to believe that water is absorbed from the digesta before they reach the abomasum (Garton, 1951; Masson & Phillipson, 1952), particular attention has been given to the volume changes that have occurred during the course of these experiments. The results show clearly that the rumen epithelium is readily permeable to water, but further work is needed to establish the direction of the flow under normal conditions. Some evidence has been found to support the contention of Sperber & Hyden, but the support so far is qualified. No evidence has been obtained to show that fatty acid absorption is affected by the ions under investigation. METHODS Sheep previously fitted with an ebonite rumen cannula were used in all experiments, and the surgical techniques for the isolation and emptying of the rumen and for the withdrawal of blood are identical with those previously described (Masson & Phillipson, 1951). Absorption of any

2 PERMEABILIT Y OF R UMEN EPITHELI UM 453 constituent was measured by the net changes occurring in the solution in the rumen and by comparing this change with the venous-arterial difference between blood leaving the rumen and carotid blood. The expression 'venous-arterial difference' is used, as the arterial value is deducted from the venous value. Sodium in blood and in aqueous solution was estimated by the method of Weinbach (1935). When appreciable concentrations of phosphate were present the trichloroacetic acid filtrate was made alkaline to phenolphthalein by the addition of dilute ammonium hydroxide. Approximately 0.5 g of powdered calcium hydroxide was then added and the solution shaken. The calcium phosphate was allowed to settle for an hour and the solution was filtered, reacidified and made up to a suitable volume. The subsequent procedures were identical with those previously described. Potassium in blood was estimated by the colorimetric method of Looney, Dyer & Worcester (1942). Aqueous solutions were diluted to bring the potassium content within the range found in blood. Chloride in blood was estimated by the open Carius method of Van Slyke (1923). Preliminary digestion was omitted when dealing with aqueous solutions. Total fatty acid in aqueous solutions was determined by steam distillation. An equal quantity of a saturated magnesium sulphate solution containing 2-5% sulphuric acid (McAnally, 1944) was added to the sample, as this prevents the appearance of chloride in the distillate. The modification of the McClendon procedure, the accuracy of which is given by Kiddle, Marshall & Phillipson (1951), was used for total volatile acid in blood. The freezing-point of rumen liquor and of blood serum was used as an indirect measure of the osmotic pressure of these fluids. The procedure described by Findlay (1932) was followed. Normally fed sheep fitted with rumen cannulae provided rumen liquor. The samples were drawn rapidly without suction, filtered rapidly through gauze and stored under liquid paraffin. Blood was withdrawn from the jugular vein by a syringe and transferred to a centrifuge tube under liquid paraffin. The serum so obtained was used for the determination of the freezing-point. The effect of loss of carbon dioxide or rumen liquor by exposure to the air was examined by determining the freezing-point after exposure and then again after re-equilibration with a mixture of 60% CO2 and 40% N2. This was a necessary precaution, as the gas phase of the rumen contains a high proportion of carbon dioxide. RESULTS The absorption of sodium and potassium acetate Two experiments in which increasing concentrations of sodium and potassium acetate were placed in the rumen showed that, so far as could be judged by the concentrations of these two ions in the blood leaving the rumen in comparison to carotid blood, no absorption occurred until the concentration in the rumen exceeded that of carotid blood. The essential data are given in Table 1. In each experiment the rumen was drained thoroughly, the initial solution of acetate was introduced and blood samples were withdrawn after approximately 15 min; a sample of the solution was withdrawn for analysis and the rumen was partially emptied. An appropriate quantity of a concentrated sodium or potassium acetate solution was added to increase the concentration, and the solution was returned to the rumen. At the end of the experiment the rumen was emptied, the residue recovered, and its volume noted. The net changes in sodium, potassium and water over the whole experiment are shown in Table 2. The sodium concentration in carotid blood remained at 238 mg/100 ml. throughout the experiment except for a slight increase during the last period, while the potassium concentration varied from 39 to 40 mg/100 ml. except

3 454 D. PARTHASARATHY AND A. T. PHILLIPSON during the last period when it increased to 43 mg/100 ml. Positive venousarterial differences occurred only when the concentration in the rumen exceeded these values. The net gain in sodium is a clear indication that sodium was passing from blood to rumen throughout most of the experiment, and the apparent absorption during the last period was small in comparison. TABLE 1. TABLE 2. The venous-arterial differences between the rumen and carotid blood for sodium and potassium compared with the concentration present in the rumen Sodium (mg/100 ml.) Potassium (mg/100 ml.) r A A- Conen. V.-A. Concn. V.-A. in rumen difference in rumen difference The absorption of sodium, potassium and water over the whole experimental period when solutions inside the rumen are increasing in strength Sodium solution Potassium solution A,Al Introduced Recovered Absorbed Introduced Recovered Absorbed Water (ml.) Base (g) (- 78 m.equiv) (36 m.equiv) The net loss in water in both experiments shows that water absorption occurred owing to the fact that the solutions in the rumen were hypotonic to blood except during the last period with the sodium solutions and the last two periods with the potassium solutions. The absorption of water, together with the passage of sodium into the rumen, is the probable explanation of the decreasing venous-arterial values in the first experiment. This may be true for the potassium solutions as well, but here a net loss of potassium was observed owing to the lower concentration of potassium in the blood. In both experiments sodium and potassium were estimated in each sample, and in both a progressively increasing concentration of the ion not originally included in the solution was found. Sodium increased to a concentration of 12 mg/100 ml. in the solution of potassium acetate, while potassium increased to a concentration of 20 mg/100 ml. in the solution of sodium acetate. A positive venous-arterial difference for total volatile acid was found whenever a solution of acetate was present in the rumen except when the weakest solution of sodium acetate was present. The venous-arterial difference, however, did not increase in a linear manner with concentration, as is shown in Fig. 1. A third experiment performed in the reverse direction is also included.

4 PERMEABILITY OF RUMEN EPITHELIUM 455 C V Acetate in rumen (g/l.) Fig. 1. The venous-arterial difference for total volatile acid between rumen and carotid blood drawn approximately 15 min after the introduction of solutions of varying strength of sodium (O or (D) or potassium (0) acetate into the rumen. The arrows indicate the order in which the solutions were introduced in the three experiments. The upper limit of the physiological concentration of acetate in the rumen is approximately 5 g/l. The absorption of sodium, potassium and chloride from rumen liquor Three litres of rumen liquor were drawn from normally fed sheep, and this material was introduced into the empty rumen and allowed to remain there for 1 hr. It was then withdrawn and 4-2 g potassium dihydrogen phosphate and 4-6 g of sodium chloride in solution were added. More rumen liquor was added to bring the volume to the same as it was previously, and the solution was returned to the rumen for another hour. Blood samples were taken at 10 and 60 min after each solution was introduced into the rumen. The complete results are given in Tables 3 and 4. The stability of the concentrations of the ions in question in the peripheral circulation allows considerable confidence to be placed upon the venous-arterial differences, and a comparison of the net gains or losses against the venous-arterial differences demonstrates the reliability of the latter measurement as a guide to whether or not absorption is occurring. This experiment shows that absorption of potassium can occur from the rumen of normally fed sheep. It has also several other interesting features. Water is lost even from normal rumen contents. It is not easy to empty the rumen completely of a solution, and we do not consider that a volume change of less than 50 ml. is significant. This is based onthe many measurements that have been made in the course of these and other experiments in which the rumen, previously emptied until only slow drips appear from the cannula, is

5 456 D. PARTHASARATHY AND A. T. PHILLIPSON washed with 31. of water or saline. The recovery is usually between 2950 and 3000 ml. During the first period there was a gain in sodium, as was to be expected from the first two experiments, and in the second period a small loss was found which again is to be expected as the concentration in the rumen exceeded that of blood. During the first period a gain in chloride was found, but an appreciable loss of chloride occurred in the second period even though its concentration was less than half that of blood. This shows that chloride can move from TABLE 3. Contents The concentrations of sodium, potassium, chloride and total volatile acids as mg/100 ml. in blood after rumen liquor has been introduced into the rumen Total fatty acid Sodium Potassium Chloride (mg acetic acid) of rumen Venous Arterial Diff. Empty Rumen liquor: After 10 min After 60 min Rumen liquor + potassium and chloride: After 10 min After 60 min (a) TABLE 4. Venous Arterial Diff. Venous Arterial Diff Rumen liquor Volume in rumen (ml.) 2750 Sodium 5-6 Potassium 3-6 Chloride 0-89 Total volatile acid 10-1 (b) Rumen liquor + NaH2PO4 + KCI Volume in rumen (ml.) 2765 Sodium 8-2 Potassium 4-7 Chloride 3-7 Total volatile acid min 60 min after intro- after introduction duction A Venous Arterial Diff The absorption of sodium, potassium, chloride and total volatile acid from rumen liquor (ph ) Concentration changes (mg/100 ml., T.V.A.* Total quantities expressed as mg acetic present (g. or ml.) acid) *T.V.A.=total volatile acids. Lost or gained (g or ml.) ml. +04 g -0-3 g g -1.lg - 85 ml. Og -004 g - 03 g g 10 min 60 min after intro- after introduction duction the rumen against a concentration gradient but not apparently until a certain concentration in the rumen is reached. These changes are clearly reflected by the blood analyses taken at 60 min and shown in Table 3. Wherever there has been a loss, the concentration in the blood leaving the rumen has been higher than in the carotid blood even though the differences are small. Conversely, net increases in the rumen are associated with measurably less of the material

6 PERMEABILITY OF RUMEN EPITHELIUM 457 in question, sodium or chloride, in the venous blood leaving the rumen than in the carotid blood. The losses of total volatile acid cannot be taken as indicative of total changes, for production of fatty acids in rumen liquor continues during the experimental periods. From the blood picture it appears as though absorption was greater during the second period than during the first, but according to the net changes this was not so. It is possible, however, either that production of fatty acid increased after the addition of the sodium phosphate and potassium chloride solutions or else that metabolism by the rumen epithelium was altered; either event would produce the blood picture observed. The influence of tonicity on absorption from the rumen It is clear from the previous experiments that considerable movement of water can occur across the rumen epitbelium. The following series of experiments were designed to see whether movement of water as such would influence the rate of absorption of ions in solution, and for this purpose potassium, chloride, acetate and propionate were chosen. In addition, experiments were designed to see whether the absorption of acetate and propionate was influenced by the presence or absence of potassium. In each experiment a comparison of three solutions was made over the period of an hour; this allowed the comparison of two variables in each experiment. Analyses of the carotid and rumen blood for the ions in question were made; samples of blood were drawn before the experiment started when only water was present in the rumen and then at the end of each period before the test solution was removed. The changes in concentration of potassium, sodium, chloride and fatty acid were determined in the rumen, and the total changes were calculated from tbe volumes introduced and recovered in the solution and in the washings. The values given for total changes are also corrected for the small quantities withdrawn in the samples taken for analysis. The inclusion of fatty acid in at least one period of each experiment had an additional value in that it served as a guide to the condition of the animal. The number of ions included in each solution was increased as the experiments progressed. The first experiment was intended to act as a guide to the quantities of sodium, chloride, potassium and acetate that could be absorbed when the solutions present were hypotonic to blood. The three 0x08 M solutions were compared. These were: (1) 0-08 M-NaCl, (2) 0026 M-NaCl M-KCI, and (3) 0026 m-nacl M-CH3COONa. The result is given in Table 5. Positive differences between the venous and arterial blood for chloride, acetate and potassium were found wherever absorption occurred, although for potassium it was so small that taken by itself it is not significant. With sodium only trivial changes were observed, but these are not indicated by the venous-

7 458 D. PARTHASARATHY AND A. T. PHILLIPSON arterial difference. Substantial quantities of chloride disappeared from the rumen when the concentration in the rumen was practically the same as that of the arterial blood, while no appreciable absorption took place when the concentration in the rumen was less than 100 mg/100 ml. In the next experiment the same concentrations of potassium and acetate were included in solutions whose molarity were: 0-165, and 0-22 M. The first two were considered to be isotonic with blood and the last hypertonic. The composition of these solutions were: (1) 0-055M-CH3COONa M-NaHCO3, (2) M-CH3COONa M-NaHCO M-KCl, and (3) M-CH3COONa M-NaHCO M-KCl. The absorption of a TABLE 5. The absorption of potassium, sodium, chloride and acetate from the rumen when 0-08 M solutions were present Concentration (mg/100 ml.) Blood Rumen contents Total lost A - A or gained Solutions Venous Arterial Difference In Out (g) Empty Potassium Empty Sodium Empty Chloride trace Empty Acetate Solution (1): M-NaCl. Solution (2): M-NaCl KCI. Solution (3): M-NaCl CH,COONa. standard concentration of acetate was compared at isotonicity with blood with and without the presence of potassium. Absorption from the same concentrations of acetate and potassium was compared using iso- and hypertonic solutions. The results given in Table 6 show no indication that the absorption of acetate is in any way influenced by the presence of potassium; neither is chloride essential, as solution one contained none. More potassium, sodium and chloride was absorbed from the hypertonic solution than from the isotonic solution, and the reverse was true for acetate. Appreciable quantities of chloride disappeared from the rumen against a concentration gradient, but sodium was only absorbed when the concentration in the rumen exceeded that of the blood. Similar experiments were done in which both acetate and propionate were added to the solutions. The first comprised a comparison of two hypotonic

8 PERMEABILITY OF RUMEN EPITHELIUM 459 (0-11 M) and one hypertonic solution (0 334 M) of the following c-mposition: (1) M-NaCl M-KCl, (2) M-CHI3COONa M-CH3CH2. COONa M-KCl, and (3) M-NaCl M-NaHCO M-KCl. The reverse comparison was made between solutions (1) and (2) to see whether the presence of fatty acid influenced the absorption of potassium, while a further comparison between (1) and (3) allowed the effect of tonicity on the TABLE 6. The influence of potassium and tonicity on the absorption of acetate from the rumen Concentration (mg/100 ml.) Blood Rumen contents Total lost A A r A or gained Solutions Venous Arterial Difference In Out (g) Empty Potassium *6 Empty Sodium trace Empty Chloride Empty Acetate Solution (1): M-CH3COONa M-NaHC08. Solution (2): M-CH3COONa M-NaHCO M-KCI. Solution (3): m-ch3coona m-nahco m-kc1. absorption of potassium to be studied. The results are given in Table 7. More potassium was absorbed from the hypertonic solution than from the hypotonic; and in each period a positive venous-arterial difference was found which agrees with the quantities absorbed. Changes in sodium were trivial until the concentration in the rumen far exceeded that in the blood in period (3). Chloride was absorbed in relation to its concentration, and the venous-arterial differences reflect the actual quantities absorbed. During period (2) chloride was absorbed against a concentration gradient. This experiment was then repeated with the difference that two isotonic solutions (0-165 M) with and without potassium were compared with a hypertonic solution (0.333 M). Acetate and propionate were present throughout. The solutions used were: (1) M-CH3COONa M-CH3CH2COONa M-NaHCO M-NaCl, (2) the same as (1) except that M-KCI was substituted for the sodium chloride, and (3) 0037 M-CH3COONa M-CH5CH2COONa + 0l11 M-NaHCO M-NaCl M-KCI. A further comparison of the absorption of fatty acid, in this experiment acetate

9 460 D. PARTHASARATHY AND A. T. PHILLIPSON and propionate with and without the presence of potassium, was made and the absorption of all three was compared between iso- and hypertonic solutions. The results given in Table 8 confirm previous experiments. The loss in potassium was greater from the hypertonic than from the isotonic solution; sodium was absorbed only when its concentration exceeded that of the blood; chloride, on the other hand, was absorbed against a concentration gradient from solutions (1) and (2). Fatty acid absorption was not greatly influenced by either the presence of potassium or by the change in tonicity. The losses or gains of potassium, sodium and chloride are all reflected by the venousarterial differences. TABLE 7. The absorption of potassium, chloride and fatty acid from hypo- and hypertonic solutions in the rumen Concentration (mg/100 ml.), ~~~~A Blood Rumen contents Total lost _ A A or gained Solutions Venous Arterial Difference In Out (g) Empty Potassium * *7 Empty Sodium trace trace *5 Empty Chloride *3 Empty Fatty acid Solution (1): m-nacl M-KCI. Solution (2): m-ch3coona M-CH3CH2COONa M-KCl. Solution (3): M-NaCl m-nahco M KC1. The effect of ph As bicarbonate was used in many of these experiments the reaction of the solutions was slightly alkaline; in order to study the influence of ph on the absorption of these ions two more experiments were performed in which the same concentrations of acetate, propionate and potassium were used and the tonicity was varied by adding sodium chloride in different strengths. The ph was adjusted to approximately 5 and maintained in that region in the rumen throughout the period by the addition of further amounts of N-HCI when necessary. The three solutions used were 0111, and M and were identical except that sodium chloride was added to increase the tonicity. The two experiments differed only in that in the first the order in which the solutions

10 PERMEABILITY OF RUMEN EPITHELIUM 461 were introduced into the rumen was that given above, while in the second this order was reversed. The hypotonic solutions consisted of M-CH3COONa, M-CH3CH2. COONa and 0055 M-KCl. To the second was added M-NaCl to bring the molarity to M, and to the third 0-22 M-NaCl to bring the molarity to 0333 M. TABLE 8. The effect of potassium and of tonicity on the absorption of fatty acid from the rumen Concentration (mg/100 ml.) Blood Rumen contents Total lost or gained Solutions Venous Arterial Difference In Out Empty (g) Potassium Empty Sodium Empty Chloride Fatty acid Empty I Solution (1): 0*037 M-CH3COONa M-CH3CH2COONa M-NaHCO M-NaCl. Solution (2): M-CH3COONa M-CH3CH2COONa m-nahco, M-KCI. Solution (3): M-CH3COONa M-CH3CH2COONa + 0l11l M-NaHCO M-NaCl m-kci. The results of the first experiment, given in Table 9, show an increase in the absorption of potassium with increased tonicity. Large quantities of chloride were absorbed, in the first period against a concentration gradient. As hydrochloric acid had to be added at intervals throughout the experiment, only the concentration of chloride in the solutions removed from the rumen are given. The rate of absorption of the fatty acids was greater than in previous experiments, which was expected from previous work. The behaviour of sodium was similar to that found in previous experiments. In this, as in all experiments, the quantities absorbed and the venous-arterial differences agree. The second experiment, performed in an identical manner in the reverse direction, gave a result that is similar in all its features. Only the total results are given in Table 10. The range of ph in these experiments was always acid; in the first the ranges of ph found for the three solutions whilst in the rumen were , and In the second they were 4-9-6'3, and

11 462 D. PARTHASARATHY AND A. T. PHILLIPSON TABLE 9. Absorption of inorganic ions and fatty acid from acidic solutions of varying tonicities in the rumen Concentration (mg/100 ml.), ~ ~ ~~A Blood Rumen contents Total lost or ra"--a_ gained Solutions Venous Arterial Difference In Out (g) Empty Potassium *3 Empty Sodium trace trace Empty Chloride P * *3 Empty Fatty acid (expressed as acetic acid) Solution (1): M-CH8COONa +0*018 M-CH3CH2COONa M-KCl. Solution (2): 0*037 m-ch3coona +0*018 m-ch3ch2coona m-kcl M-NaCl. Solution (3): 0*037 m-ch8coona m-ch3ch2coona+0*055 m-kcl+0022 M-NaCl. TABLE 10. The total quantities of inorganic ions and fatty acids lost or gained from acidic solutions of varying tonicity Fatty acid (expressed Water Potassium Sodium Chloride as acetic acid) Solution (ml.) (g) (g) (g) (g) 1 (0*33M) * (0'16 M) trace -P (0.11M) trace The effect of mercuric chloride From these experiments there was reason to suppose that the absorption of chloride, as it is absorbed against a concentration gradient, and possibly also acetate, since there is apparently more efficient absorption at lower concentrations than at higher ones, is an expression of the activity of the epithelial cells lining the rumen. Ingraham & Visscher (1936) have shown that small concentrations of mercuric chloride will inhibit absorption of chloride from the ileum of the dog. Mercuric chloride is a general protoplasmic poison and has no known specificity as an inhibitor, but, as none of the known inhibitors is likely to affect the uptake of chloride, this agent was investigated in two experiments. In the first experiment a solution containing sodium chloride, bicarbonate, acetate and the acid phosphate giving a total molarity of was used in the first period of an hour. Chloride was absorbed against a concentration gradient to the extent of 0-6 g, the initial concentration being

12 PERMEABILITY OF RUMEN EPITHELIUM mg/100 ml. Acetate was absorbed to the extent of 0 9 g from an initial concentration of 229 mg/100 ml., and 120 ml. of water disappeared. An identical solution containing in addition M-mercuric chloride was used in the second period. Chloride in the rumen increased by 01 g, acetate absorption was reduced to 0 3 g and only 40 ml. of water disappeared. No inorganic phosphorus was absorbed. In the second experiment a hypertonic solution was used to provide optimal conditions for the absorption of chloride. It consisted of potassium chloride, sodium chloride, sodium bicarbonate, sodium acetate and sodium acid phosphate, giving a total molarity of 0-33 M. In the first period 2-6 g of sodium was absorbed from an initial concentration of 660 mg/100 ml.; 0-7 g of potassium from an initial concentration of 206 mg/100 ml.; 3-1 g of chloride from a concentration of 678 mg/100 ml.; and 2-1 g of acetate from a concentration of 332 mg/100 ml. 360 ml. of water were gained. In the second period from an identical solution containing M-mercuric chloride, 2-3 g sodium, 0-8 g potassium, 1-7 g chloride and 1-2 g acetate were absorbed and the gain in volume was 150 ml. A trace of inorganic phosphorus, 0-1 g, was lost during both periods from a concentration of 74 mg/100 ml. These experiments permit us to divide the constituents studied into two classes: (1) chloride and acetate whose absorption was reduced or stopped by mercuric chloride, and (2) potassium and sodium whose absorption was unchanged or only slightly affected by this agent. Volume changes were reduced no matter in which direction they occurred. The absorption of water It was mentioned earlier that volume changes of less than 50 ml. were not considered significant. The procedure for emptying the rumen has been standardized so that, provided care is taken to see that any fluid that enters the thoracic part of the oesophagus and the reticulum can drain downwards to the cannula, recoveries of 2950 ml. or more can be made from 3000 ml. An earlier experiment performed for another purpose showed that water could be absorbed from the rumen. The reticulo-omasal orifice was ligated and the rumen was emptied and washed. The organ remained empty during the experiment and after 3 hr, at post-mortem, it was found that the interior, instead of being moist, was completely dry, and the small residues of fibre left inside after the washing were so dry that they could readily be blown about in contrast to their habit of sticking to the walls of the organ. Particular attention was paid in all these experiments to volume changes that occurred and these are set out in Table 11. In addition to the changes given in Table 11 substantial losses occurred during the first two experiments when increasing concentrations of sodium and potassium acetate were introduced into the rumen which were for the most part hypotonic to blood

13 464 D. PARTHASARATHY AND A. T. PHILLIPSON (Table 2). From normal rumen liquor 210 ml. were lost, and 85 ml. were lost from liquors to which KC1 and NaH2PO4 had been added (Table 4). The volume changes found experimentally were large enough to make it interesting to study the osmotic relationship between the blood and the rumen contents of normally feeding sheep. Samples of rumen liquor withdrawn at intervals throughout the day were treated as previously described and the freezing-point obtained. TABLE 11. The volume changes (ml.) in the rumen over periods of 1 hr when solutions of different tonicity are present Strength of solution in rumen (M) TABLE 12. A of rumen liquor and serum of normally fed sheep Sheep no. Time Fed on hay, oats and linseed meal Rumen liquor 9 a.m a.m P.m p.m p.m Serum Fed on hay and oats Rumen liquor 9 a.m a.m P.m p.m p.m Serum X591 Equilibration of rumen liquor with a mixture of 60% C02 and 40% N2 after removing coarse solid particles increased the value of A by only C. For this reason the results have not been corrected for loss of carbon dioxide due to exposure to the air as the error involved is so small. Table 12 gives the results and also the values obtained for the serum of the sheep concerned. These results show that the osmotic pressure of rumen contents are never greatly different from that of blood serum, but the differences are large enough to allow considerable movement of water. The daily variation shows in six out of the seven of the series a decrease in the value of A immediately after feeding. With the richer of the two rations this is followed by a steady rise so

14 PERMEABILITY OF RUMEN EPITHELIUM 465 that the value becomes greater than that of the serum. When the ration of hay and oats was fed without linseed meal, which is a protein-rich foodstuff, the value of A was nearly always less than that of the serum. DISCUSSION These studies on the absorption of sodium, potassium, chloride and water from the rumen support the evidence put forward by Sperber & Hyden (1952) to show that the rumen epithelium is a selective membrane, although our results differ from theirs in that we have not found that sodium is absorbed against a concentration gradient and chloride only when concentrations of 135 mg/100 ml. or more are present in the rumen. The venous-arterial difference has proved a reliable guide to whether or not absorption is occurring, but these values can alter with time after the introduction of the test solution. Consequently they can only be accepted as a qualitative guide for inorganic ions. A closer relationship exists, however, for acetate when the values for the venous-arterial differences are far larger. The quantities of potassium disappearing were influenced by the tonicity of the solution; losses of from 0-2 to 0 4 g/hr were observed from isotonic solutions when the concentration present was from 133 to 220 mg/100 ml. Greater absorption was found from hypertonic solutions that were slightly alkaline due to the inclusion of bicarbonate when losses of g/hr occurred. This increased rate of absorption, however, did not occur where hypertonic but acid solutions were placed in the rumen. Losses of g/hr of sodium were found from concentrations in the rumen of from 360 to 385 mg/100 ml., while losses of 1 0-2*4 g occurred from solutions containing mg/100 ml. Regular losses of chloride were found against a concentration gradient. In quantity these ranged from 0 3 to 0-6 g/hr. They occurred, however, from solutions that were 135 mg/100 ml. or greater; otherwise chloride in the rumen increased when concentrations of less than 100 mg/100 ml. were present. Under normal circumstances the chloride concentration in the rumen is less than 100 mg/100 ml. and may be as low as 25 mg/100 ml. (Masson & Phillipson, 1952; Parthasarathy, 1952 a). From these experiments it seems as though the rate of entry of chloride into the rumen equals its rate of passage to the -blood when the concentration in the rumen is between 100 and 135 mg/100 ml. This critical level may well be lower in normal unanaesthetized sheep. Absorp- -tion of chloride against a concentration gradient has been recognized for a long time in the dog; recently, Visscher, Fetcher, Carr, Gregor, Bushy & Barker (1944) have shown that isotopic chlorine moves in both directions across the mucosa of the ileum but at a greater rate from the lumen of the gut to the blood so that a net loss is found. It appears as though the same principle holds good for the rumen. PH. CXXI. 30

15 466 D. PARTHASARATHY AND A. T. PHILLIPSON No account has as yet been taken of the influence of the divalent ions, calcium and magnesium. Garton (1951) found concentrations in the rumen from 7 to 20 mg/100 ml. for soluble magnesium and from 10 to 21 mg/100 ml. for soluble calcium in the rumen liquor of normally fed sheep. Potassium, sodium and ammonia are the three principal bases in the rumen, and it is clear from these experiments that potassium is steadily absorbed, while McDonald (1948) has shown that ammonia is also readily absorbed. Of the anions, fatty acids and chloride are absorbed and there is little doubt that carbon dioxide and bicarbonate are also absorbed. Inorganic phosphate is the only anion which penetrates the epithelium in very small quantities (Sperber & Hyden, 1952; Parthasarathy, Garton & Phillipson, 1952). The large venous-arterial difference between rumen and carotid blood found in the normally fed but anaesthetized sheep for sodium by Parthasarathy (1952b) and the losses of sodium recorded by Sperber & Hyden (1952) from a Pavlov pouch of the rumen are good reasons to believe that failure to find absorption against a concentration gradient in these experiments is due to the condition of the experiment, but the reason for this is far from clear, for it should be noted that absorption from normal rumen contents did not occur. Similarly, the fact that absorption of chloride against a concentration gradient did not occur until the concentration was 135 mg/100 ml. needs explanation. We have found no relationship between the absorption of acetate and propionate and the presence or absence of potassium; the only factors that influenced the absorption of fatty acids were lowering the ph-a well-known effect-and the introduction of M-mercuric chloride which decreased absorption of chloride, water and fatty acid. There is, however, a clear relationship between the concentration of acetate in the rumen and the venous-arterial differences, and the fact that the relationship is not linear is interesting, for it suggests that absorption is more efficient from weak solutions than from stronger ones. The losses of acetate in vitro due to metabolism by rumen epithelium are small compared with those for butyrate and propionate (Pennington, 1952), so that a relation can be expected between these two sets of values. Poisoning ofthe rumen epithelium by exposing it to 0X002 M-mercuric chloride inhibited absorption of the anions, acetate and chloride, while absorption of the cations, potassium and sodium, proceeded unhindered. The absorption of chloride against a concentration gradient may mean either an active participation of a chemical mechanism of the epithelial cells themselves or a movement of ions according to a difference in electrical potential across the membrane, which in turn can only occur as a result of selective activity of the epithelial cells. The concentration of acetate in the blood is so small that a favourable concentration gradient always exists, yet the fact that mercuric chloride depresses the quantity absorbed in a similar manner to acetate, together with the fact that absorption from low concentrations appears to be

16 PERMEABILITY OF RUMEN EPITHELIUM 467 more efficient than from higher concentrations, are good reasons for believing that the absorption of acetate in common with chloride is influenced by the activity of the epithelial cells lining the rumen. Absorption of potassium, however, was unaffected by the presence of mercuric chloride and the effect on sodium was very slight. Mercuric chloride, therefore, in spite of its apparent lack of specificity, has successfully divided the ions into two groups: those whose absorption is stopped or greatly reduced and those which are unaffected. There is no reason to postulate any mechanism for the absorption of potassium beyond that of diffusion from a higher to a lower concentration; if any specific mechanism is concerned with the absorption ofsodium it appears to be different to that concerned with the absorption of chloride. Absorption of water from the rumen under the experimental conditions indicates that absorption occurs normally, for the figures for A of the rumen contents are often slightly hypotonic to the blood serum. Measurement of the freezing-point of complex biological fluids is not easy, and the probability is that owing to supercooling the actual values for A recorded are slightly lower than the true value. Aldred (1940) measured the osmotic pressure of sheep's blood taken from normally fed animals and found a range equivalent to *169 M-NaCl with an average of M. These values correspond to a depression of freezing-point of ' C with an average of C. These are greater than the values for A found for the serum of our sheep. Admittedly serum, having no fibrinogen, can be expected to have a slightly lower osmotic pressure than whole blood, but as the total osmotic pressure exerted by plasma colloids is not greater than 0-3 % of the total this factor will be negligible. In any event, the higher the osmotic pressure of blood the more favourable are conditions for the absorption of water. The rumen liquor always had a lower freezing-point early in the morning than later in the day; with sheep feeding on hay, oats and linseed meal the contents became distinctly hypertonic to the serum, but when they were feeding on hay and oats alone it was nearly always slightly hypotonic to blood. The calculated values for A for the strength of solution used in these experiments, namely 011, 0-165, 0-22, 0-33 M, assuming they consist entirely of NaCl and dissociation is 100% are: 0 409, 0-614, and respectively; this shows that only one of our solutions, namely, M, is within the physiological range of tonicity, and that it is very slightly hypotonic when compared to Aldred's figure of A for sheep's blood. Within an hour, however, losses of ml. occurred. It is interesting that Visscher et al. (1944), who studied the movement of water and deuterium from the ileum of the dog, found that, although movement of water, generally speaking, followed the osmotic gradients, movement of water from gut to blood occurred when the osmotic activity gradient was zero or negative. The values for A found in the three sheep feeding on hay and oats and in one animal on the richer ration were 30-2

17 ,468 D. PARTHASARATHY AND A. T. PHILLIPSON always lower than 0*614, and periods occurred in the remaining three sheep on the richer ration in which the value for A was lower than 0X614. For this reason we conclude that water absorption occurs from the rumen of the normally feeding sheep. Whether movement of water occurs in the reverse direction is not so clear; the hypertonic solutions used in these experiments were well outside the normal range, and although the rumen liquor on the richer diet certainly rose above Aldred's value of for blood, we have no data to show whether that can cause a significant movement of water into the rumen, although it is theoretically possible; but theoretical considerations are not necessarily correct with solutions that are slightly hypertonic, according to the observations of Visscher et al. (1944). Movement of water into the rumen in the single experiment in which an 0x22 M solution was employed was only 30 ml. The calculated value for A for this solution is which is greater than any of the values found in normal sheep. SUMMARY 1. The venous-arterial differences between rumen and carotid blood for potassium, sodium and chloride were found to be reliable indications of whether absorption was occurring from the rumen in significant quantities or not. 2. Sodium and potassium were absorbed from the rumen when their concentrations exceeded that in the blood. Passage in the reverse direction occurred when their respective concentrations were lower than those of blood. 3. Chloride was absorbed from the rumen against a concentration gradient provided the concentration was 135 mg/100 ml. or over. At concentrations lower than this, net gains in chloride in the rumen were found. 4. The presence or absence of potassium did not affect the absorption of acetate or of propionate. 5. The presence of M-mercuric chloride inhibited the absorption of chloride and acetate but did not affect the absorption of potassium and sodium. 6. Absorption of water from solutions of M strength or less occurred. The volume of solutions of 0-33 M increased when they were introduced into the rumen. 7. The osmotic relations between normal rumen contents and blood are such that absorption of water from the rumen under normal conditions is possible. The authors wish to thank Mr L. E. Vowles and Miss Mildred Wilson for their assistance in these experiments.

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