Walther(3), who used animals with permanent fistulae of the pancreatic

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1 OBSERVATIONS ON PANCREATIC SECRETION. BY G. V. ANREP, JOAN L. LUSH AND M. GRACE PALMER. (From the Institute of Physiology, University College, London.) THE experiments of Terroine (1, 2) and his collaborators, showed that in protracted secretion of the pancreatic juice the concentration of all three enzymes underwent a considerable diminution. This diminution, however, did not affect the three enzymes to the same extent; while the decrease in concentration of the proteoclastic enzyme and of amylase was gradual, the concentration of lipase showed a steep decline and practically disappeared by the end of the experiment. These observations are supported by the experiments made in Pavlov's laboratory by Walther(3), who used animals with permanent fistulae of the pancreatic duct; he also found that the concentration of the three enzymes does not run parallel, that of lipase always decreasing most rapidly. Walther's experiments, however, cannot be accepted without reservation, since they were made before the discovery of enterokinase and of the reinforcing action of bile upon lipase. Also, in the experiments of Terroine, Lalou and others, although enterokinase was used, the lipase was not reinforced with bile salts. If T err o in e's observations upon the rapid diminution of concentration of lipase are correct, it would seem either that the amount of lipase stored in the pancreas is much less than that of the other two enzymes, or possibly that the cells are less efficient in the production of lipase. Subsequent experiments performed in Pavlov's(4) laboratory upon animals with permanent or temporary fistulae under widely different conditions, such as digestion of different food-stuffs in the one case, and administration of acids and stimulation of the vagus in the other, showed complete parallelism in the changes in concentration of all three enzymes; but no long experiments where secretion was protracted by injection of secretin were reported. It might, therefore, be concluded that the difference in the result can be explained on the grounds that secretin has a specific effect on the pancreas causing a quick exhaustion of the lipase without affecting the other two enzymes. In view of the discrepancy between the results we undertook to

2 PANCREATIC SECRETION. 435 re-investigate the question, using more accurately standardised methods than those used by Lalou and others. Methods. All experiments were performed on dogs ansesthetised with chloralose (-1 gm. per kg.). Secretin was prepared in the ordinary way, either from the animal's own intestine or from that of other dogs. The injection of secretin was made from a burette connected with the jugular vein. By regulating the inflow of secretin the rate of pancreatic secretion was kept as constant as possible. The juice was collected in test-tubes under paraffin and occasionally samples of blood were taken from the femoral artery; the blood was also collected under paraffin. The following analyses were made: In the blood. Carbon dioxide content by Van Slyke's method. ph of whole blood by Dale and Evans' method. In the pancreatic juice. Carbon dioxide content and ph by the same methods. Total alkalinity by means of addition of excess of standard acid, and back titration with standard alkali, using methyl orange as indicator. Amylase, by means of the Achromic Point method. Trypsin, by means of S6 r ens on's formalin method, after activation by addition of enterokinase. The method employed for determination of lipase needs to be described in detail, since we are not aware that it has been used before. Since it seems generally agreed that the concentrations of trypsin and amylase show the same rate of diminution in protracted secretion, in only a few experiments were both determined. In all experiments the changes in concentration of lipase were compared with those of amylase. The methods which are generally used for estimation of lipolytic activity are based on titration with a standard alkali of the fatty acids produced in the digestion mixture. Usually the pancreatic juice, with or without the addition of bile, is added to a watery solution of a lower ester or to an emulsified fat. In the latter case, the degree of emulsification should be well standardised, but such a procedure is difficult and never certain. In both cases the digestion mixture quickly reaches a very high H-ion concentration, which is detrimental to the enzyme, since the optimal H-ion concentration for pancreatic lipase is about 10-. This obvious source of error was eliminated in our experiments by the use of buffered solutions which allowed only a small change in ph as digestion proceeded. In a preliminary experiment we were able to show that in two digestion mixtures of the same initial ph, one buffered as described below and the other unbuffered, the degree of hydrolysis in

3 436 G. V. ANREP, J. L. LUSH AND M. G. PALMER. the buffered solution was six times as great as that in the unbuffered; in the former case, under the conditions of our experiments, about 25 p.c. of the available ester was hydrolysed when the ph shifted from 7-8 to 7-0. The method adopted was, shortly, as follows. Glycerol triacetate was used as substrate. A strongly buffered phosphate solution of ph 7-8 or 8-0 was used to stabilise the H-ion concentration. A few drops of a suitable indicator (usually phenol red) were added to the digestion mixture and the time taken to produce equal changes in the ph of the mixture, as shown by the indicator, was used as a measure of the lipoclastic activity. The shift in the ph was traced only as far as ph 7-0, further change being considered too remote from the optimal ph. The changes were best determined by the use of the Dale and Evans' comparator method. In most of the experiments this method was checked by back titration, using phenolphthalein as indicator. Both methods always gave entirely comparable results. It is obvious that the use of a buffered solution in this method is intended not only to keep the reaction of the medium close to the optimal, but also to show the rate of hydrolysis. The addition of natural bile to the digestion mixture is incompatible with any colorimetric method because of the strong colouration of the medium with bile pigments. We therefore tried other substances in place of the bile. Saponin and caprylic alcohol were tried first, on account of their effect on surface tension, similar to that of bile. They were found to be without any reinforcing effect upon lipase. Synthetic bile salts were used next and sodium glycocholate proved to have as strong an action as bile itself (Exp. 1). After trying various proportions of glycerol triacetate, buffer solution, bile salts and pancreatic juice, the following mixture was found to give the best results: 5 c.c. 1 % glycerol triacetate (freshly prepared). 5 c.c. phosphate buffer solution as used in Dale and Evans' method. -5 c.c. -3 % sodium glycocholate. -3 c.c. pancreatic juice. 8 drops *02 % phenol red or of another suitable indicator. Average samples of pancreatic juice gave a change from ph 7-8 to ph 7-0 within about an hour, and we found that under these conditions only about 25 p.c. of the available glycerol triacetate was hydrolysed. Control tubes containing the same mixture without the addition of pancreatic juice showed only a negligible amount of hydrolysis.

4 PANCREATIC SECRETION. 437 Time taken to produce Exp. 1. change from ph 8-0-pH 7 0 Digestion mixture containing 5 c.c. 1 % glycerol triacetate mins. 5 c.c. phosphate buffer + *5 c.c. of *3 % saponin c.c. pancreatic juice + 3 drops of caprylic alcohol 185,, 8 drops phenol red + *5 c.c. of *3 % sodium glycocholate 45 It is evident from this experiment that the reinforcing action of bile salts is not due simply to surface tension phenomena causing a greater dispersion of the lipase, but to a specific chemical action of the bile salts. Exp. 2 is an example of the group of experiments in which variations in the rate of hydrolysis obtained by increasing the concentration of the pancreatic juice in the digestion mixture were studied, and a comparison made between the rate in unbuffered and in buffered digestion mixtures. c.c. of Time taken to produce a change from ph 8-0 to pancreatic, A Exp. 2. juice ph 7-8 ph 7-6 ph 7-4 ph 7.2 ph 7-0 Usual mixture but with water iia place of the buffer solution 03 Changed to below ph 6-0 in 5 mins. Usual mixture Do Do Do Changed to about 7 9 in 300 mins. It is obvious from this experiment that in the unbuffered mixture the H-ion quickly reaches a value so high that it would hinder further action of the lipase. Changes in enzyme activity of pancreatic juice in protracted secretion. In the seventeen experiments performed it was quite clear that all three enzymes suffered a proportionate diminution in concentration. The following two experiments illustrate the point. Exp. 3. Amount of juice secreted 5 c.c. 10 c.c. 20 c.c. 30 c.c. 40 c.c. Lipase; time taken to effect a change from ph mine. 65 m. 50 m. 80 m. 145 m. Amylase; time taken to reach the achromic point 60 secs. 90 S. 80 s. 130 s. 300 s. Exp. 4. Sample Amount of juice secreted 25 c.c. 50 c.c 100 c.c. 125 c.c. 150 c.c. 175 c.c. Lipase; time taken to effect achangefromph mins. 80 m. 100 m. 110 m. 250 m. 350 m. Amylase; time taken to reach the achromic point 70 secs. 77 s. 90 S. 122 s. 200 s. 285 s. In the same experiment the tryptic activity showed a diminution of the following degree. To produce a digestion equivalent to 10 c.c. N/10 NaOH Sample Took 7 hrs. 9j hrs. 15 hrsi

5 438 G. V. ANREP, J. L. LUSH AND M. G. PALMER. The change in concentration of the three enzymes does not show a greater rate of diminution in the lipase than in the other two enzymes. These results confirm the results of Pavlov's school, and are in contradiction to the views held by Terroine. There is no need to give further examples since the results of all our experiments were concordant on this point. It is well known that preparations of secretin made in exactly the same way excite a different amount of secretion in different animals. It has generally been attributed to differences in the store of secretin which the animal's intestine contained before the experiment, or to slight variations in the process of extraction. On several occasions the same preparation of secretin was tested on different animals, and on the other hand several preparations of secretin were tried on the same animal. It was found without any doubt that the most important factor in producing a good secretion lay in the preparation of the secretin; but at the same time, on several occasions we found that in some animals secretion was provoked in large amounts even with small doses of secretin, while in others the same secretin caused only a scanty secretion of juice. In none of our experiments was any relation observed between the amount of juice secreted and the rate of diminution of enzyme concentration. In some experiments the enzymes showed a considerable diminution with a total secretion of only 12 c.c. juice; in others, the same diminution was obtained only after a secretion of over 175 c.c. Thus the rate of diminution of enzyme concentration does not depend on the amount of juice secreted. Moreover, little evidence was obtained to show that the rate of diminution of enzyme concentration depends on the rate of secretion. If, however, two successive small samples secreted at a different rate were analysed, it was found in some experiments that the juice secreted most rapidly was richer in enzymes than that secreted at a slower rate. To show this effect, the experiment has to be performed on a pancreas that secretes well, otherwise it is masked by the progressive diminution in the enzyme concentration. These experiments extend the Heidenhain phenomenon to the pancreatic gland. Exp. 5 illustrates this point. Exp. 5. Sample Amount of juice collected c.c. 6 c.c. 6 c.c. Time taketi for juice to be secreted mins. 40 m. 36 m. Lipase; time taken to produce a change from ph 7-7-7*0 50 mins. 28 m. 45 m. The change in the rate of secretion from 90 mins. to 40 mins. was accompanied by an increase in the concentration of the enzymes. The

6 PANCREATIC SECRETION. third sample showed the effect of progressive diminution in enzyme concentration. However, these results are not so clear nor so easily obtained as in the case of the salivary gland. The rate of diminution of enzyme concentration seems to depend primarily upon the fitness of the gland to produce the pancreatic juice. A gland secreting little, produces a juice whose concentration of enzymes is very low after only a few c.c. have been secreted, while a gland secreting well produces a juice with a high concentration of enzymes, even after a secretion of over 100 c.c. juice. It occurred to us that both the difference in the amount of juice secreted as well as the rate of diminution of the concentration of enzymes in the juice could be explained most easily by the state of exhaustion of the gland before the experiment. This explanation, however, proved incorrect, since an injection of pilocarpine caused a very considerable increase in the concentration of enzymes (Exp. 6). Moreover, it has been shown before by Babkin, Rubashkin and Savich(5), that even large amounts of juice secreted in response to injection of secretin do not cause exhaustion of the gland as judged from histological observations. Lipase; time taken to produce a Exp. 6. change from ph Sample at end of secretion of 40 c.c. 140 wins. 439 First sample after injection of pilocarpine 15 mins. This experiment shows that the store of the gland was not depleted by the secretion of the juice. From Bayliss and Starling's observations (6), it is known that the alkalinity of the juice gradually falls as secretion proceeds, and this is partly due to removal of available alkalies from the blood, and partly to injections of the slightly acid solutions of secretin. It is evident that an animal which has more available base would maintain the alkaline reaction of the juice for a longer time. Taking into consideration these observations we tried to find whether the concentration of the enzyme stands in relation to the titratable alkalinity of the juice, or the H-ion concentration of the blood.- The following experiment shows that there is no such relation. The titratable alkalinity in Exp. 7 does not change much. The C02 content of the juice falls progressively, to rise after each injection of Na2CO2. The ph and C02 content of the blood increase after these injections, but the progressive fall in the concentration of lipase does not seem to depend in any way upon the changes in the alkalinity of the pancreatic juice or blood. The experiment shows that when a rest of

7 440 G. V. ANREP, J. L. LUSH AND M. G. PALMER. Exp. 7. Amount Titratable C'02 content C02 content Lipase; of juice Haemo- alkalinity of juice of blood ph of change secreted globin in in vols. in vols. arterial from in c.c. % N/10H2SO4 % blood ph *153N mins * * * * ,, Intravenous injection of 60 c.c. of 10 % sodium carbonate * ,, * '77 210, Rest for 45 mins.; injection of 60 c.c. of 10 % sodium carbonate * *78 85,, * , 45 mins. was allowed, the concentration of lipase increased sharply. Since, during the period of rest, 60 c.c. of Na2CO3 were injected slowly into the blood, we had to make sure that the latter was not the cause of the rise in concentration of the enzymes. In several experiments resting periods were interposed with periods of injection of secretin. It was found that after every resting period the juice was much more concentrated. Since it is known that the vagus nerve has a potent influence upon the excretion of enzymes, we tried to find whether this augmentation in enzyme concentration after rest is in any way dependent on the integrity of the vagus nerve. The experiments showed that the enzyme concentration in the juice increased after rest, even when the vagi were cut after administration of atropine or both. Stimulation of the vagus with Faradic current did not, in our hands, cause any larger increase in the enzyme concentration than was caused by rest alone. In this respect our results differed from those of S av i ch (5), who showed a definite increase in concentration of the enzymes during the period of stimulation of the vagus. His experiments Exp. 8. Lipase; tirne taken to produce a clhange from Titratable alkalinity ph of juice 2 20rnins. *141N,, *134 pi, *132 Amount of juice in c.c Vagi cut; 15 mins. rest Rest for 30 mins Vagus stimulated for 30 mins ,, V,, joy *122 *124 *106 *108 *111 *105 *111 *111 *120

8 PANCREATIC SECRETION. were conducted on spinal animals. Our experiments also differ from those of Babkin(7), who obtained the effect of the vagus on animals anaesthetised in a similar way to ours but with both splanchnic nerves cut. However, it is evident that the augmentation in the concentration of the enzymes, which was observed in our case, was independent of the vagus mechanism. That the vagus nerve does not regulate the re-establishment of a gland during the periods of rest is shown also by the experiments in which both vagi were cut during the period of secretion. If this is done at the very beginning of secretion, without discarding the first few cubic centimetres of juice, it causes a considerable diminution in the concentration of enzymes, which proves once more an old observation of Pavlov. If, however, the vagi are cut after secretion has proceeded for some time, it has no effect on the concentration of enzymes. This observation supports the experiments of Savich and others. On looking through our experiments we find that the only possible explanation of the facts described in this communication is found in the difference of the general state of the experimental animal. Those animals which showed only a gradual and incomplete recovery of bloodpressure after injection of secretin, secreted badly and the enzyme concentration was low. Animals which kept their blood-pressure high to the end of the experiment were of the type which secreted much juice and in which the enzyme concentration fell slowly. Along with Babkin's experiments this shows once more the importance of a good blood supply to the pancreatic gland. 441 During the periods of rest the blood-pressure improved considerably, and this seems to be the only explanation of the apparent recovery of the gland. The above explanation is corroborated by the observation that after injections of histamine, secretin causes a much smaller secretion of pancreatic juice and the juice contains considerably less enzymes (Exp. 9). Exp. 9. Dog, 8 kilos. Four injections of 1 c.c. of secretin each caused a secretion of 1-2; 1-5; 1-3; and 1-4 c.c. of juice. Between each injection the blood-pressure was allowed to recover to normal (120 mm. Hg). After an injection of 0-2 mgm. of ergamine diphosphate the blood-pressure fell to 40 mm. and did not recover for a long time. Injections of the'same dose of secretin caused a flow of juice of only 0-2; 0 35; 0 1; and 0-12 c.c. After an interval of one hour the bloodpressure had risen to 80 mm. and the secretion improved. The concentration of enzymes was very considerably less in the juice which was collected during the period of low blood-pressure. When the blood-pressure and the secretion recovered the concentration of the enzymes rose nearly to the previous level.

9 442 G. V. ANREP, J. L. LUSH AND M. G. PALMER. CONCLUSIONS. 1. In protracted secretion of pancreatic juice maintained at a constant rate by continuous injection of secretin, all three enzymes show a parallel diminution in their concentration. 2. Contrary to the view of Terroine and Lalou, the lipase does not show a more rapid fall in concentration than the other two enzymes. 3. This diminution in concentration of enzymes is not a true exhaustion of the gland as is shown by experiments with pilocarpine. 4. The lipase was estimated in these experiments by a new standardised method which should prove of value in experiments dealing with any lipoclastic activity. 5. The progressive fall in the concentration of enzymes does not depend upon the alkalinity of the juice, the alkalinity of the blood, or on the absolute amount of juice secreted; neither can it be explained by changes in the rate of secretion. 6. After a short rest the pancreatic gland secretes a juice with a higher concentration of enzymes. The effect of rest is independent of the vagus mechanism, and occurs also after administration of atropine and after section of both vagi. 7. The explanation is advanced that the changes in the concentration of the enzymes depend upon the general condition of the animal and the blood supply to the gland. The expenses of this work were defrayed out of a grant from the Medical Research Council held by one of us (G.V.A.). REFERENCES. (1) Lalou. C. R. Acad. d. Sci (2) Morel and Terroine. C. R. Soc. Biol (3) Walther. Arch. des Sci. Biol (4) Pavlov. "The work of the Digestive Glands." Trans.: W. H. Thompson, (London: Griffin.) (5) Babkin, Rubashkin and Savich. Arch. mik. Anat (6) Bayliss and Starling. This Jourr (7) Babkin. "Die aussere Sekretion d. Verdaiiungsdrusen," (Berlin: Springer.)