when the concentration of magnesium in the lumen of the gut was greatly sheep are known to be permeable to 28Mg [Care, 1960] although on the basis of

Transcription

1 FACTORS INFLUENCING THE SECRETION AND ABSORPTION OF CALCIUM AND MAGNESIUM IN THE SMALL INTESTINE OF THE SHEEP. By D. SCOTT. From The Department of Physiology, The Rowett Research Institute, Bucksburn, Aberdeen. (Received for publication 23rd January 1965) Secretion and absorption of calcium and magnesium were studied in sheep prepared with loops of the jejunum and ileum that could be isolated temporarily from the flow of digesta. The secretion of the isolated loops was discharged in an irregular manner; distension of the loop caused an increase in the rate of secretion but neither a period of feeding nor the level of food intake produced any recogniable effect. By comparison, the flow of digesta in the upper jejunum usually increased following a meal and showed clear dependence upon food intake. Both calcium and magnesium were absorbed from inorganic solutions placed in the isolated loops. Net absorption of calcium depended on the concentration of calcium in the solution introduced but no such relationship could be established in the case of magnesium. The introduction of inorganic solutions into the isolated loops was accompanied by a considerable reduction of the electrical potential gradient normally present between the intestinal lumen and blood. Glucose restored the potential gradient but the concentrations required were much greater than those normally found in digesta. It is suggested that no more than a small fraction of the calcium and magnesium absorbed from the sheep's intestine is absorbed by simple diffusion of free ion. STEWART and Moodie [1956] demonstrated that magnesium was absorbed from all regions of the alimentary tract of the sheep from rumen to caecum when the concentration of magnesium in the lumen of the gut was greatly increased following oral dosing or local injection of magnesium salts. Absorption occurred at the greatest rate and from the lowest concentration in the small intestine. When the concentration of magnesium in the gut contents was kept at normal levels, Field [196] was able to show that the middle third of the small intestine was the main site of absorption of magnesium in the sheep. The reticulo-rumen and the duodenum of the sheep are known to be permeable to 28Mg [Care, 196] although on the basis of net absorption there is no significant uptake of magnesium in these regions in the sheep [Care, 196; Storry, 196] or in the calf [Smith, 1959 a]. Smith [1959 b, 1962] has shown that from 26 to 4 per cent of the dietary magnesium was absorbed by the large intestine of milk-fed calves of from 2 to 4 weeks of age although by 6-12 weeks this function was lost. Absorption of magnesium is also believed to occur in the large intestine of the rabbit [Aikawa, 1959]. Moore and Tyler [1955] reported that the absorption of calcium from the small intestine of the pig was greatest in the first quarter. In rabbits [Thomas et al., 1954], chickens [Gershoff and Hegsted, 1956] and rats 312

2 Secretion and Absorption in the Sheep Intestine [Nicolaysen, 1937] the small intestine is also believed to be the main site for calcium absorption, particularly the region immediately caudal to the stomach [Harrison and Harrison, 1951; Nicolaysen, 1951; Schacter and Rosen, 1959; Schacter et al., 196]. The principal sites of calcium absorption in the ruminant are not well defined. This paper provides information on factors which control the secretion and absorption of calcium and magnesium in the intestine of sheep. METHODS Animal8. - The sheep used were five Blackface ewes and one Greyface wether (166) between 12 and 18 months of age and averaging 45 kg. liveweight. Preparation of Intestinal Loops. - Pentobarbitone injected intravenously was used to anaesthetie the sheep. The intestine was transected at two points about 6-7 cm. apart. The ends were closed and cannulated using cannulae of the type described by Ash [1962] or modifications of those previously described by Vaughan-Williams M31 3 FIG. 1. The preparation of intestinal loops by the exterioriation of the flow of food material. [1954]. The cannule were then exterioried through stab wounds and the first and second cannulee and the third and fourth were joined by tubing. A loop of intestine was thus made in continuity with the remaining intestine by way of two external connexions through which all the digesta entering and leaving it had to pass (fig. 1). In sheep 395, 1366 and 166 the prepared loop of intestine was in the upper jejunum, beginning about 1 cm. caudal to the ileac flexure. In sheep 1378 the loop was placed roughly two-thirds of the distance between the pylorus and ileo-caecal junction. In sheep 1373 and 1375 the loop was placed in the terminal ileum finishing 1 cm. before the caecum. Recovery following surgery was in most cases uncomplicated and the flow of digesta was restored within hr. and full appetite within about 4 days. No animal was used for experimental work until 6-8 weeks after the operation and the sheep used in the absorption experiments served useful experimental lives of 9-18 months. Only very occasionally was the flow of digesta interrupted through blockage.

3 314 Post-mortem examination of sheep 395, and length of loops. In these animals there was no indication of alteration in the appearance or sie of the intestine included in theloop compared with the adjacent intestine. In sheep 1375 the lumen was slightly enlarged but the mucosa appeared to be normal and 1373 confirmed the position Measurement of Secretion and Flow of Digesta. - At the beginning of each experiment the loop was isolated from the normal flow of digesta and washed with warm NaCl solution (3 m-osmole/kg.) until the washings were clear. The loop was drained and 45-6 min. were allowed to elapse to permit recovery from the washing procedure. During this period the digesta flowing from cannula1 was collected, its volume was recorded and it was then returned to cannula 4 over approximately the same time period as taken for its collection. The total flow of digesta from cannula 1 and that of the loop secretion from cannula 3 were noted for each successive 1 min. period, the ph of the loop secretion being recorded immediately. After two consecutive 3 min. periods the sheep were fed and collection of digesta and loop secretion was continued for a five-hour period after feeding. Diets and Feeding Regime. - The first group of experiments were conducted over three consecutive 7-day periods. The sheep were fed at 9.3 A.M. and 5 P.M. and the food was usually consumed within 5-1 min. During the first period the sheep were fed 4 g. grass cubes at each feed and experiments on flow and secretion were performed on day 3 and day 5. At 5 P.M. on day 5 intake was changed to 2 g. per feed and experiments conducted on days 3 and 5 of period 2 whereafter intake was increased to 55 g. per feed and experiments conducted on days 3 and 5 of period 3. Six further experiments with the same protocol were conducted using chopped dried grass on three intakes of 2, 4 and 5 g. per feed. The animals were again fed twice daily and the food usually eaten within 5-15 min. Measurement of Absorption. - Before the experiment cannulae 1 and 4 were joined with a piece of glass or polythene tubing so that the flow of digesta by-passed the loop. The loop was then washed with warm NaCl solution (3 m-osmole/kg.) until the washings were clear and during this operation care was taken to ensure that the tissue was not unduly distended. Extensions to cannulse 2 and 3 were then made using suitable lengths of polythene tubing which terminated in glass funnels held above the animal. The loop was thoroughly drained and a known volume of solution containing calcium and magnesium was introduced into it through cannula 2. The solutions used in these experiments contained calcium, magnesium, sodium and potassium chlorides, their tonicity being 3 m-osmole/kg. and their ph In most solutions calcium and magnesium were present in equal concentrations although in a few magnesium was omitted altogether. At the end of the absorption period the solution was recovered into a measuring cylinder from cannula 3, recovery being assisted by gently blowing down cannula 2. The loop was then washed three times using 1, 5 and 5 ml. of the NaCl solution respectively, at 37 C. A fresh experimental solution was then introduced; solutions differing in composition being introduced randomly. The net absorption of calcium and magnesium during the experimental period was calculated from the quantity introduced into the loops and from that recovered at the end of the period including the wash solutions. Appropriate corrections were made for drainage errors. A + ve sign is used to denote net absorption of calcium and magnesium as measured by a loss from the experimental solution. In some experiments solutions of calcium and magnesium were perfused into cannula 2 and collected from cannula 3 of an isolated jejunal loop. The rate of perfusion was controlled so as to be similar to the rate of flow of digesta for the particular diet and level of feeding. At the end of the experiment the loop was drained and washed as described above. Scott Chemical Methods. - Calcium and magnesium were estimated by a modification

4 Secretion and Absorption in the Sheep Intestine 315 of the method of Wilson [1955] using ethylene diaminetetracetic acid. The standard error of the mean of duplicate estimations for calcium was ±8 mg. when taking aliquots containing 1--4 mg. of calcium, and recoveries from inorganic solutions containing magnesium varied from 99. to 11-8 per cent. The standard error of the mean of duplicate estimations for magnesium was +5 mg. when taking an aliquot containing mg. of magnesium while recoveries from inorganic solutions containing calcium varied from 98-7 to 12 per cent. Sodium and potassium were estimated by flame photometry (Evans Electroselenium Model A), chloride by the method of Sanderson [1952], total C2 by the method of Van Slyke and Neill [1924] and osmotic pressure using the Fiske Osmometer (Model G). Glucose was estimated by the method of Somogyi [1952]. Potential Measurements. - The bio-electrical potentials between the intestinal lumen and the blood stream were recorded using calomel half-cells and a Cambridge ph meter on the mv scale. Connexions to the animal were made using saturated KCI-agar bridges. Bridges of 1-2- mm. bore were used to connect with the intestinal contents and a bridge of 5 mm. bore was threaded through a hypodermic needle into the jugular vein to make contact with the blood. This procedure is essentially the same as that described by Dobson [1959]. Potential measurements were made in sheep 395, 166 and 1378 which were fitted with isolated loops of intestine. In addition the potential was measured at a number of sites in the gut using seventeen other conscious sheep fitted with one or more established intestinal cannule through which bridges could be inserted. RESULTS The Secretion of Intestinal Juice and Flow of Digesta in the Small Intestine The secretion of the jejunal loops was discharged from cannula 3 in an irregular manner and the volumes collected on these occasions were variable. Neither the period of feeding included in the experiment nor the level of food intake were associated with any recogniable change in the rate of secretion of intestinal juice in the isolated loop (fig. 2). By comparison with secretion of intestinal juice the flow of digesta usually increased following a meal and showed clear dependence upon food intake (fig. 2). In addition, the digesta flow was greater on the chopped grass ration than on the grass cubes although the rations provided the same dry matter intake. Water intake was higher on the grass-cube diet than on chopped grass, the difference being greater at the higher food intakes. Only four experiments were conducted on the flow of digesta in the terminal ileum, for unlike the jejunum, where the flow was more or less continuous, flow in the ileum was very infrequent, considerable volumes being propelled during relatively short periods interspersed with long periods of inactivity. Under these conditions 5-6 hr. observations did not reveal any relationship between flow and food intake such as was found in the jejunum. Samples of ileal secretion collected from the distal end of the ileal loops showed that secretion of intestinal juice was not influenced by feeding or by the flow of digesta through adjacent areas of the gut. The volumes collected were small and their discharge from cannula 3 was irregular. The average composition of both jejunal and ileal secretions is given in Table I. The juice collected comprised two components, one a clear or

5 316 Scott a~i1 u in * *- A c 1 P3-4!. WI.:.. - m 8 o 8 (IC C> 'la 1-.- : ioi.1 2 h V L.I Si

6 Secretion and Absorption in the Sheep Intestine 317 x w Q C) C. H o C E- o P- es t CO CD P O C CO w ( CO E41 pq 2 P4 ~1 1 l r-4 16 eq o =~ - P- co - v) CO cio 1 m.,- HEe s tdn L O O 2 p. v )2o.,.- 16 t- I eq E-4 CO O 4 C o 6 (DO CO CD? Crl~ p4 ȮQ C- CO I 2 1- C~ C I+,-4(= 141 H 41 E4 C o *;- CD6 CO ( 2 D CD - E-1 P4 E1 v v * *. I -,4 1 (19 - g S.- ; ; V 26 rj2 14 $:4+) 2) 4

7 318 Scott slightly opalescent fluid, varying from colourless to pale yellow; the other, a yellow-coloured solid material which appeared to be largely composed of cellular debris and comprised less than.5 per cent of the total weight. Both secretions had a characteristically fishy smell. a 2.. oo D I o. os e;4 ;, o -1 Ileal loops mep :,1 Co Concn. tmoles/l a c 5a A B S b to. o c? jejunal loops sheep o *&6 'a 4 a X ad Ca Concn. m.moies,/. FIG. 3. The net absorption of calcium from isolated ileal and jejunal loops. The concentration of calcium in the solutions is plotted (a) as the initial concentration and (b) as a mean of the initial and final concentrations with a correction for change in volume. The Effects of Distension upon Secretion of Intestinal Juice Collections were made from the distal end of an isolated jejunal loop in sheep 395. The first 5 cm. of the loop were stimulated by distending a short balloon to a pressure of 1-15 mm. Hg. This gave rise to an increase in the rate of flow of secretion from cannula 3 (Table II). Distension of the whole jejunal loop of sheep 1378 by inflating it with an

8 Secretion and Absorption in the Sheep Intestine air pressure of 8-1 mm. Hg pressure produced a similar response. The composition of these secretions was very similar to those of the corresponding unstimulated secretions (Table II). The Absorption of Calcium and Magnesium Both calcium and magnesium were absorbed from inorganic solutions placed in jejunal and ileal loops. Where the solutions changed little in volume, as was usually the case for the ileal loops, and the concentration of calcium was less than 2 m-mole/l. then the net uptake of calcium was dependent upon the concentration of calcium in the experimental solution, uptake increasing with increasing concentration. This relationship was to some extent obscured when volume changes were large as in many experiments with jejunal loops. In such cases the change in calcium concentration is likely to have arisen both TABLE III. THE ABSORPTION OF CALCIUM IN THE JEJUNUM OF THE SHEEP (Loop 395) Net uptake Ca Initial concentration Volume change r- - (m.mole/l.) (ml.) (y-mole/5 ml./hr.) (Percentage) X X 29X as a result of dilution with intestinal secretion and absorption, and it was found that the scatter of results was reduced if calcium uptake were plotted against a mean value for calcium concentration. This was arbitrarily taken as the average of the initial concentration, corrected for volume change, and the final concentration (fig. 3). A similar interpretation of the results was obtained by plotting the geometric average of the initial and final concentrations. Net absorption of calcium from inorganic solutions placed in isolated jejunal loops gave similar results to those for the ileal loops, uptake increasing as the concentration of calcium was increased up to 15-2 m-mole/l. (fig. 3). Above this concentration uptake of calcium was variable (Table III). Analysis of the supernatant fluid of digesta for these sheep revealed that calcium was normally present at concentrations varying from m.mole/l. and magnesium from m-mole/l. By comparison with calcium no conclusive relationship was established between net absorption of magnesium and the concentration of magnesium in the experimental solutions (fig. 4). Absorption of magnesium in the jejunal loops was in most cases positive although variable. In the ileal loops 5 per cent of the experiments gave negative values for net absorption of magnesium and in many cases a negative result was obtained with a solution which in an earlier experiment gave a positive result.

9 32 Scott When solutions containing equivalent quantities of calcium and magnesium were introduced into a jejunal loop, uptake of calcium from a given initial concentration was found to depend upon the time during which the solution was left in the loop, whereas uptake of magnesium from the same solution was not (Table IV). aoo r a b a 2 o A.oI 55 S * o % 4 III 2 2. o a o an -tool 3 -ao. Mg Concn. m.molevl. Ieal loops * sp I, a b ' r a Iu ot 2 tooi. a * *A *2 to 4 4' OS la 6 DO n Jejunal loops sheep G -1, Mg Concn mmoles/l. FIG. 4. The net absorption of magnesium from isolated ileal and jejunal loops. The concentrations of magnesium in the solutions is plotted (a) as the initial concentration and (b) as the mean of the initial and final concentration, with a correction for change in volume. Perfusion Experiments. - Calcium was absorbed from inorganic solutions perfused through the isolated loop but a relationship between net uptake and concentration was not evident and uptakes were comparatively small relative to the quantity of calcium perfused. Net uptake of magnesium from the solutions was very irregular (Table V). In subsequent experiments whole jejunal digesta were centrifuged at 2 g for 3 min. and the supernatant fluid was removed. When this

10 Secretion and Absorption in the Sheep Intestine 321 supernatanit was perfused through the isolated loop uptakes of calcium and magnesium were variable and in some cases negative. When fixed amounts of this supernatant fluid were mixed with the inorganic solutions containing calcium and magnesium uptakes of these elements were found to be no greater than from the inorganic solutions alone and there was no obvious relationship between the net uptakes and the respective concentrations of calcium and magnesium (Table V). The Electrical Potential Gradient. - The potential difference between the intestinal lumen and the jugular vein under various conditions is shown in fig. 5. When digesta was present in the jejunal loops of sheep 395 and 166 there was a potential gradient between the intestinal lumen and the jugular vein TABLE IV. THE ABSORPTION OF CALCIUM AND MAGNESIUM IN THE JEJUNUM AS DETERMINED BY THE LENGTH OF EXPERIMENTAL PERIOD (LooP 395) Length Initial concentration Net uptake of Volume - -'--A---II period change Ca Mg Ca Mg Expt. (min.) (ml.) (m.mole/l.) (,u.mole/5 ml./hr.) * X X67 2* * l8 13* * of 1-15 mv, blood positive. Once the loop had been washed out and filled with an inorganic solution the potential gradient was much reduced. In many cases the potential fell rapidly within 5-1 min. of introducing the solution into the loop to values of 1-2 mv and occasionally the blood became 1-2 mv negative to the lumen. The introduction of the supernatant fluid from the digesta into the loop once again raised the potential to values near those found when normal digesta was present. This supernatant had a similar inorganic composition to the experimental solutions which suggested that some of the soluble organic substances present in the digesta were necessary for the maintenance of the potential. A few of the substances normally present in the digesta were examined and the results are shown in fig. 5 while the composition of the -solutions used is given in Table VI. The concentration of total volatile fatty acids in the rumen is around 95 mm while in the jejunum this concentration is less than 5 mm [Badawy et al., 1958]. The substitution of the sodium salts of acetic, propionic and butyric acids for part of the sodium chloride in the experimental solutions VOL. L, NO

11 ro Co Co Co Co EH 322 :.-4-4Q O C 4N-4 t ot Cmr ll i X6 r ; I I I tbb d C uuc C od - NCO11 1 to o o M. '. Co C) Cd N - O 1± I - s C; C; 1 l l r- = C lo = O O O bid Co 4-4 " cc CD-s Scott O to o M X s wco CS- --Co> - - H7 --o.o.h Co Co ObO M"- C a) + r X es IH H X - o -o> s s c H o oooo Co. - S d o ) -q ol c e SX Q t: S *1 - -m OCO N 11 : o6 o6 O 6O - '* - co o xt 16 < : ;- 16 C; Cs : : : GI-.f *4- k7. -4 P- o o ~ :~... CO.- -C C CO COc IC. cc ḞZ CO. +- t-4 o ot1o 1 ID D - ll - *..... n :3 o ZZt o

12 Secretion and Absorption in the Sheep Intestine 323 at concentrations of both 1 mm and 95 mm were found to give potential gradients much below those recorded between blood and digesta flowing past the loop. In experiments with sheep 395 the substitution of 6 mm glucose for part of the sodium chloride in an inorganic solution was accompanied by a rise in the potential gradient to above the values recorded between blood and digesta. A similar response was subsequently obtained in the jejunal loops of sheep 2 - [ * o~ OO Solution Isolated oo o o o I loop 3 o it ooooo{ cdgsalo o o oo OOo 22 e S of Digesto lc o wa va a ooj. W loop lo wer 1. j go t m o i 2 34S Time (hr) FIG. 5. The electrical potential gradient between blood and solutions in an isolated jejunal loop in sheep 395. The composition of the solutions used is given in Table VI. 166 and 1378, using solutions containing 2-6 mm-glucose. Concentrations of 1-15 mm-glucose in solutions with or without volatile fatty acids gave potential gradients similar to those found when normal digesta was present in the ioops, while lower concentrations of glucose gave much lower potential differences. Solutions containing 2 mm-mannitol or 6 mm-mannose, both non-metabolites, were found to give potenitial gradients very much below those recorded between blood and digesta, whereas a solution containing 2 mm-galactose gave a potential gradient comparable to that recorded for a 2 mm-glucose solution. In two subsequent experiments with sheep 166 the introduction of a solution containing 2 mm-glucose was initially accompanied by an elevated potential gradient which then showed a progressive fall. Analyses of small samples of the solution in the loop taken as the experiment proceeded showed that the glucose was rapidly absorbed (fig. 6).

13 324 Scott DISCUSSION The secretion of intestinal juice by the isolated loops did not appear to be stimulated by feeding or by the rate of flow of intestinal digesta. The sheep appears to be similar to the dog in this respect for Boldyreff [194] showed little or no spontaneous secretion in the fasting dog but rather a small periodic flow of secretion which Babkin and Ishikawa [1912] suggested relates to intestinal motor activity. This conclusion was supported by Komarov PD mu,v digesta sf [ *ol loop 4 *_ proximal ',dso I quantity vv time (mgns) FIG. 6. The relationship between the potential gradient and the disappearance of glucose from a jejunal loop (loop 166). [1924] who failed to show any response to feeding in Thiry or Thiry-Vella loops in the jejunum of the dog and showed that the small periodic secretion could be inhibited by atropine. On the other hand, the results of the distension experiments indicate that local distension of the loops can accelerate the secretion of intestinal juice although this had little effect on composition. Similar findings have been reported for the dog following the introduction of an inflated balloon or a sponge rubber bolus which excited the propulsive motility of loops and led to increased secretion [Gregory, 1962]. Earlier than this many workers studying the composition of intestinal secretions have frequently used collection techniques which have either deliberately or unavoidably involved some mechanical stimulation, a point seldom commented upon by the authors [Babkin, 1928; Nasset and Parry, 1934; de Beer et al., 1935] but referred to by Gregory [1962]. The results of the distension experiments also show that when a loop of

14 Secretion and Absorption in the Sheep Intestine 325 intestine is isolated from the flow of digesta the rate of secretion of intestinal juice is likely to be influenced by the volume of the solution introduced into the loop as well as by the chemical composition of the solutions used. In the present experiments it was therefore desirable to standardie procedure in order to minimie variation in endogenous secretion of calcium and magnesium arising from this source. Both calcium and magnesium were absorbed from solutions placed in isolated loops in the jejunum and the ileum of conscious sheep. For calcium a relationship between absorption and concentration was clearly visible, as it is in dogs [Cramer and Dueck, 1962] and it persisted up to 15-2 m.mole/l. which are close to the upper concentrations of calcium founld in normal digesta. A number of workers have suggested that calcium and magnesium may compete in a common transport process since absorption of calcium by everted sacs of rat, rabbit and guinea-pig intestine [Schacter and Rosen, 1959], by slices of rat small intestine [Schacter et al., 196] and by Thiry- Vella loops of the jejunum in the dog [Cramer and Dueck, 1962] was slightly reduced if magnesium were included in the mucosal solutions. In the present experiments there was no marked difference between net absorption of calcium from solutions in which magnesium was present or absent so that any competition between calcium and magnesium in absorption in the sheep is sufficiently small to be concealed in the variability which exists between experiments. By comparison with calcium, no relationship was found between magnesium uptake and its concentration in the experimental solutions and indeed in miany experiments, particularly in the ileal loops, there was a net secretion rather than an absorption of magnesium. The introduction of inorganic solutions into isolated loops of the sheep small intestine was found to be accompanied by a great reduction of the electrical potential gradient normally present between the intestinal lumen and the blood. The potential gradient across the gut wall is believed to be associated with the active transport of sodium and the diffusion of potassium into the blood [Dobson, 1956; Dobson and Phillipson, 1958; Harrison et al., 1964]. The inorganic solutions used in the present experiments, however, contained these ions at much the same concentrations as in normal digesta. Chloride was at or below the concentration found in digesta, and inorganic phosphate was absent, but one would expect this to cause an increase in potential. The inclusion of fatty acids or a protein digest in the test solutions had no effect on the potential so that the presence or absence of such organic ions seems not to be concerned in the fall in potential in these studies. The finding that glucose could restore the potential gradient lends support to the conclusions of Barry et at. [1964] that the maintenance of the potential gradient demands an adequate source of metaboliable energy and that the digesta may normally supply at least part of this requirement. Analyses of the supernatant fluid of digesta gave values for total reducing sugar of only 1-2 mm which is less than the concentration found in blood, 2-3 mm, so that in the sheep absorption of sugar must occur against a concentration gradient

15 326 Scott as has been found in the rat [Barry et al., 1961; Hart and Nissim, 1964]. By comparison concentrations of glucose between 1-2 mm were needed to maintain the electrical potential gradient between blood and inorganic solutions at the level found between blood and digesta. Previous workers have also found it necessary to employ concentrations of glucose much above physiological levels in order to obtain a response in electrical potential in the intestine. Cooperstein and Brockman [1959] reported that a Ringer solution containing 7.2 mm-glucose could not sustain a potential gradient in Thiry-Vella loops in the small intestine of the dog while Clarkson et al. [1961] found that the potential difference across midjejunal segments of the rat intestine in Ringer solution could be increased from the range mv up to mv using a concentration of 14.4 mmglucose in the mucosal solution. Barry et al. [1964] have recorded potential differences in vivo in the rat small intestine up to 12 mv, using concentrations of glucose up to 54 mm and the potential gradient both in everted sacs and in in vivo segments of intestine depended upon the concentration of glucose present in the mucosal solution. The need for abnormally high concentrations of glucose in in vitro preparations denied a normal blood supply can perhaps be justified, but their need in intact animals is difficult to understand and the present results give no further information on this point. If a substantial fraction of the calcium and magnesium absorbed from the gut passes through the intestinal wall by simple diffusion as free ions, then a reduction in the sie of the electrical potential gradient opposing their absorption is likely to be important. Storry [1961 a and b] showed that the acid secretions of the abomasum cause almost all of the calcium and magnesium of the abomasal contents to be converted to a form able to pass through a collodion membrane, whereas the proportions of duodenal calcium and magnesium which were ultrafiltrable were small and decreased further as ph increased down the small intestine. If the absorption of calcium from the intestine of the sheep does depend upon diffusion of the free ion, then at equilibrium for this ion the concentration difference must be associated with an equilibrium potential (E) the magnitude of which is given by the relationship E = RT ln[ca]g/[ca]p 2F where R is the gas constant, T the absolute temperature, F Faraday's Constant and [Ca]g and [Ca]p the concentrations of free ionic calcium in the gut contents and plasma respectively. A simnilar relationship may be expressed for magnesium using the appropriate concentrations for [Mg]g and [Mg]p. The concentration of ionic calcium in the plasma of ruminants is around 2.7 m.equiv/l. [Godden and Duckworth, 1935] while that of magnesium is around m.equiv/l. [Walser, 1961]. In Table VII a comparison has been made between the concentrations of ultrafiltrable calcium and magnesium found in the gut contents of five sheep by Storry [1961 a and b] and the minimum concentrations of free ionic calcium and magnesium which

16 Secretion and Absorption in the Sheep Intestine E- q 6* -4 C'e -1 P E- g oz H om Q-1 r X P4 + ~o s C k P- ;.4.;oQ - O o -H> -H -H:-H H -H - o )k cc a (N (N-c -H -H -H -H -H -H O 1 = 6 ~- c- t%t: d4 CqP ṯ~4 (2 x o -ld + 4 P- v 9D. 4d. 4 ad E 5 ;- O.C P4! p A~~o 4 PR 2 - Z o 'F'F -5 r-4 k C) P4 a, O,: O a O P. 9 --,. p.:) 4 -"-,

17 328 Scott would be necessary for simple diffusion against the observed potential gradient at various sites in the gut. This Table indicates that the potential gradients found in the reticulorumen and the abomasum are normally too great to permit any net absorption of calcium and magnesium ions by simple diffusion. In the small intestine also, wherein the majority of calcium absorption is believed to occur, the potential gradient is usually sufficiently large to prevent simple diffusion of the free ion into the blood although it must be borne in mind that the concentrations of ultrafiltrable magnesium found by Storry [1961 b] may overestimate the concentrations of free ion actually present. It therefore seems unlikely that more than a small fraction of the calcium and magnesium absorbed from the sheep's small intestine is absorbed by simple diffusion of the free ion. This is borne out by the fact that no more calcium and magnesium were absorbed from inorganic solutions that did not support the normal potential difference between intestinal lumen and blood than were absorbed from solutions containing the supernatant fluid of digesta which did sustain the potential. The results of the potential experiments using isolated loops illustrate, however, the need to establish carefully-defined criteria of normality in these and similar preparations before any conclusions may be referred to the whole animal. ACKNOWLEDGMENTS The author wishes to express his thanks to Professor A. T. Phillipson and Mr. R. W. Ash who prepared the isolated loops and gave helpful advice. Skilled technical assistance provided by Mr. A. Ingram is also gratefully acknowledged. REFERENCES AIKAWA, J. K. (1959). Proc. Soc. exp. Biol., N.Y., 1, 293. ASH, R. W. (1962). Animal Prod. 4, 39. BABKIN, B. P. (1928). Die Aussere Sekretion der Verdauungsdrctsen, 2nd Ed. Berlin: J. Springer. BABKIN, B. P. and ISHIKAWA, H. (1912). PfliUg. Arch. ge8. Phy8iol. 147, 335. BADAWY, A. M., CAMPBELL, R. M., CUTHBERTSON, D. P. and MACKIE, W. S. (1958). Brit. J. Nutrit. 12, 384. BARRY, R. J. C., MATTHEWS, J., SMYTH, D. H. and WRIGHT, E. M. (1961). J. Physiol. 161, 17P. BARRY, R. J. C., DIKSTEIN, S., MATTHEWS, J., SMYTH, D. H. and WRIGHT, E. M. (1964). J. Phy8iol. 171, 316. BOLDYREFF, W. N. (194). Thesis: St. Petersburg. Cited by R. A. Gregory, in Secretory Mechanisms of the Gastro-intestinal Tract, 1962, 1st Ed., p London: Arnold. CARE, A. D. (196). British Veterinary Association. Report of Conference on Hypomagnesnmnia, p. 34. London. CLARKSON, T. W., CROSS, A. C. and TOOLE, S. R. (1961). Nature, Lond. 191, 51. COOPERSTEIN, I. L. and BROCKMAN, S. K. (1959). J. clin. Inve8t. 38, 435. CRAMER, C. F. and DUECK, J. (1962). Amer. J. Phy8iol. 22, 161. DE BEER, E. J., JOHNSON, C. G. and WILSON, D. W. (1935). J. biol. Chem. 18, 113.

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