ON THE EFFECT OF BILE UPON THE HYDROLYSIS OF ESTERS BY PANCREATIC JUICE. BY A. S. LOEVENHART AND C. G. SOUDER. (From the Laboratory of Physiological Chemistry of the Johns Hopkins University.) (Receivedfor publication, November 13, 1906.) The part played by the bile in the digestion and absorption of fats has long been a subject of discussion. It has been proved conclusively that the bile greatly promotes the absorption of fats and that in its absence fat absorption is much impaired. Although various views on fat absorption have been held by different investigators, all supposed that the bile aids the process by acting as a solvent for the fatty acids liberated by the action of lipase and by assisting in the emulsification of the neutral fat. Hewlett * has recently found that the pancreatic juice as secreted possesses comparatively little activity toward the esters of the lower fatty acids. He holds that the principle function of the bile is to render the lipase more active. He discusses briefly the mechanism by which the bile exercises this activating effect and concludes that someconstituent of the bile acts as a zymo-exciter or accelerator for the lipase, but dismisses as improbable the view that the bile activates a lipase zymogen. Hewlett arrived at this conclusion by using triacetin instead of the higher fats. Triacetin is quite soluble in water, and, moreorer, the products of its hydrolysis mix with water in all proportions. Hence, if bile is capable of accelerating the action of pancreatic juice on triacetin, its action in this case certainly cannot be explained by the solvent power of the bile on the zymolyte or on the prod- ucts of the reaction. Hewlett found that bile greatly accelerates the action of pancreatic juice on triacetin. Thus, in the ex- periments which he quotes the bile increased the hydrolysis twenty-six times in the first hour, while at the end of twenty- four hours the hydrolysis was one and one-half times as great as in the absence of bile. Similarly the hydrolysis of ethyl butyrate was accelerated seven to sixteen times by f Johns Hopkins Hospital Bdletin, xvi, p. 20, 1905. 415 by guest on February 19, 2020 http://www.jbc.org/ Downloaded from
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ON THE EFFECT OF BILE UPON THE HYDROLYSIS OF ESTERS BY PANCREATIC JUICE.
BY A. S. LOEVENHART AND C. G. SOUDER. (From the Laboratory of Physiological Chemistry of the Johns Hopkins
University.) (Received for publication, November 13, 1906.)
The part played by the bile in the digestion and absorption of fats has long been a subject of discussion. It has been proved conclusively that the bile greatly promotes the absorption of fats and that in its absence fat absorption is much impaired. Although various views on fat absorption have been held by different investigators, all supposed that the bile aids the process by acting as a solvent for the fatty acids liberated by the action of lipase and by assisting in the emulsification of the neutral fat. Hewlett * has recently found that the pancreatic juice as secreted possesses comparatively little activity toward the esters of the lower fatty acids. He holds that the principle function of the bile is to render the lipase more active. He discusses briefly the mechanism by which the bile exercises this activating effect and concludes that some constituent of the bile acts as a zymo-exciter or accelerator for the lipase, but dismisses as improbable the view that the bile activates a lipase zymogen. Hewlett arrived at this conclusion by using triacetin instead of the higher fats. Triacetin is quite soluble in water, and, moreorer, the products of its hydrolysis mix with water in all proportions. Hence, if bile is capable of accelerating the action of pancreatic juice on triacetin, its action in this case certainly cannot be explained by the solvent power of the bile on the zymolyte or on the prod- ucts of the reaction. Hewlett found that bile greatly accelerates the action of pancreatic juice on triacetin. Thus, in the ex- periments which he quotes the bile increased the hydrolysis twenty-six times in the first hour, while at the end of twenty- four hours the hydrolysis was one and one-half times as great as in the absence of bile. Similarly the hydrolysis of ethyl butyrate was accelerated seven to sixteen times by
f Johns Hopkins Hospital Bdletin, xvi, p. 20, 1905.
the addition of bile. This very important contribution could only have been made by using such a water-soluble ester as triacetin. Hewlett sought to determine which constituent of the bile is responsible for this acceleration. He found that commercial lecithin is capable of accelerating the action of pancreatic juice to the same extent that the bile does. He found that a commercial preparation of the bile salts also accelerates the action of the pancreatic juice on the ester but as the bile salts are purified, their accelerating action decreases. He concluded that the accelerating action of the bile salts is probably due to a contamination with lecithin. The generally accepted view has been that the bile salts are the important constituents of the bile in assisting in the absorption of fats.’ Strecker2 first called attention to the fact that the bile salts increase the solubility of the higher fatty acids in water, and the experiments of Marcet 3 showed the solubility of fatty acids in bile and the emulsification of neutral fat by this mixture. Hence the bile salts have been looked upon as the active constituents of the bile in fat absorption. Martin and Williams 4 found that the bile salts accelerate the action of pancreatic disastase, and they attribute the accelerating action of the bile in this process to the bile salts.
We have found that results obtained in the action of lipase on one ester cannot be inferred to hold when another ester is used. Hence it becomes necessary to see whether the observa- tions of Hewlett regarding the accelerating action of lecithin will hold when the higher fats are used. We have determined quantitatively the activity of pancreatic juice on several esters, including olive oil, in the gresence and absence of bile salts, lecithin, and bile. In doing this we have had two objects in view: (I) to determine, if possible, which constituent of the bile under physiological conditions accelerates the action of pan- creatic juice; (2) to determine whether the hydrolysis of the various esters is affected in the same way and to the same extent by these substances. Since the publication of a preliminary
1 Howell, A Text-book of Physiology, 1905, p. 7x5. *Ann. d. &em., Ixv, p. I, 1848.
1 Pmt. of the Roy. Sot., ix, p. 306, 1858. ’ Ibid., XIV, p. 358, 1889.
communication by one of us 1 on the part played by the bile salts in the hydrolysis of amyl salicylate by liver extract, two articles have appeared on the subject. Von Fiirth and Schiite2 in a preliminary communication state that Plattner’s bile salts possess the full activity of the bile in accelerating the hydrolysis of fat by pancreatic juice. They find, furthermore, that pure sodium glycocholate behaves similarly and that sodium cholate is also very active. They conclude therefore that the power of the bile 60 accelerate this process resides in the cholic acid complex of the bile salts. No data are given in this communica- tion Magnus has found that the bile salts synthetically prepared, accelerate the action of pancreatic juice on olive oil.
Experiwtestal Afelhods. The pancreatic juice was obtained from dogs by establishing a temporary fistula and injecting secretin. The bile was also collected in most cases, and it is interesting to note in this connection that the volumes of pan- creatic juice and of bile collected in each experiment were usually practically equal. This would indicate that in the intestine the digesting fluid probably consists of about equal volumes of bile and pancreatic juice. The biliary canula was never forced as high up as the cystic duct. In some cases the bile was ob- tained from the gall bladder directly. The titrations were carried out in the manner described on p. 3gg of the preceding article. The indicators were used as described in the preceding article, p. 399. Toluene was always used as the antiseptic. The usual blanks were carried through, and all necessary correc- tions for acidity or alkalinity have been made in the data which follow. The results are recorded in the number of cubic cen- timeters of & alkali required to neutralize the acid produced by the enzymic hydrolysis of the ester. The esters investigated and the quantities used unless otherwise stated were as follows: ethyl acetate--o.1747 gram, ethyl propionate-o.2082 gram, ethyl butyrate-0.23 gram, diacetin-o. I 7 I 4 gram, triacetin- 0.1497 gram, and olive oil-o.3 gram, The reason for using the esters in these quantities is given on page 398 of the preceding
1 Amer. Jown. of Physiol., xv, p, xxvii, 1906. 0 Centralbl. f. Physiol., xx, p, 47, 1906. a Zeitschr, f. physiol. Chem., xlviii, p. 376, 1906.
article. The duration of the experiments and the tempera- ture are given in comlection with each series. We have studied the effect of bile salts, lecithin, mixtures of bile salts and lecithin, and bile on the hydrolysis of the above mentioned esters by the juice. The bile salts were prepared from ox bile and were purified by solution and reprecipitation and finally by extracting for several days with ether in a Sohxlet extractor. A large quantity was smelted with the usual oxidation mixture and showed only a trace of phosphorus. The lecithin used was prepared by the method of Roaf and Edie l and was employed in a 10 per cent. aqueous suspension. The bile was obtained from dogs, and in most cases from the animal that yielded the pan- creatic juice of the experiment. In order to compare the bile with bile salts at various concent.rations. the bile was assumed to contain 5 per cent. of bile salts and was diluted accordingly. Thus to compare bile with a 0.1 per cent. solution of the bile salts, the bile was diluted with 49 volumes of water. This will be designated “ bile -bile salts 0.x per cent, ” etc. In the tables that follow, the concentration of the bile salts and Iecithin in the total mixture is given and not the concentration of the solution or suspension added. Thus in speaking of the action of 0.1 per cent. bile salts we mean that the bile salts were present in the total mixture in this concentration.
Expwimental Results. We have been able to confirm the observations of Hewlett that lecithin accelerates the action of pancreatic juice on the lower esters, and we have found that it also accelerates the action of the juice on olive oil. We have found that the bile salts also greatly accelerate the action of pancreatic juice on all of the esters investigated. In order to bring out the accelerating action on the hydrolysis of the various esters, it is necessary to employ the bile salts in different concen- trations. Thus for the lower esters the bile salts should be used in a concentration of from 0.1~ to 0.05 per cent. In these concentrations they do not accelerate the hydrolysis of olive oil. When this latter ester is employed, the bile salts should be used in a concentration of from 2 to 4 per cent. All of these facts are brought out in the following tables.
1 Thompson-Yates and Johnston Lab. Rep., vi (I), p. 201, 1905.
SERIES 1. Duration of experiments, 16 hours, Temperature, 38O C. One C.C. of pancreatic juice was used in each experiment and this was diluted to 2.1 C.C. in every case.
T
“a Alkali w equired. C.C.
--
3.2 6.65 6.75 9.05 9.30
10.25 -. --__
Per cent. of Accelet-
stion.
i-i -5 13 -.. _--
SERIES 2. Duration of experiments, 18 hours. Temperature, 3Q” C. One C.C. of pancreatic juice was used in each experiment and this was diluted to 3 C.C. with water or lecithin suspension. -..------7 I------
Per cent. of Acceleration.
SERIES 3. Duration of experiments, 16 hours. Temperature, 3S” C. Similar in all respects to Series 2.
I -- I ---..-.----I
SERIES 4. Duration of experiments, 19 hours. Temperature 40° C.. Two C.C. of pancreatic juice were used in each experiment and this was diluted to 3 C.C.
SERIES 5. Duration of experiments, 19 hours. Temperature, 35O C. One C.C. of pancreatic juice was used in each experiment and this was diluted in each case to 5 C.C.
Ester. Bile I I Salts.
2 Alkali Required.
per cent. C.C.
Bt>yl bu;yrate ‘I ‘I
Diacetin I‘ Triacetin ‘I
I‘ Olive oil “ I,
“ ‘1 I _-..--._-_- .-_.-_- ---- ~-- L----
-_--
Shares 6. Duration-of experiments, 22 hours. Temperature, 38” C.. One C.C. of pancreatic juice was used in each experiment and this was. diluted in each case to 6 C.C.
We have selected the foregoing experime:lts because they show the widest variations in the values which we have found. The pancreatic juice from different dogs does not give the same results. It will be seen in the tables that both lecithin and the
bile salts accelerate the action of the juice on olive oil and on the lower esters. The effect of the bile salts varies greatly with the concentration. Thus in a concentration of 0.1 per cent. the bile salts greatly accelerate the action on the lower esters and this acceleration decreases with increasing concentration. In a concentration of 0.8 to 4 per cent. they invariably inhibited the action of the juice on triacetin (Series 5). fn Series 6, 0.1 per cent. bile salts inhibited the action on triacetin. This is oc- casionally seen. In Series I, the bile salts, o,os per cent., cause practically no acceleration, while in Series 4 (0.1 per cent. bile salts) an acceleration of I 70 per cent. in the hydrolysis of triacetin is noted. We have uniformly found that pancreatic juice without the addition of any accelerator is from 3 to 4 times as active on triacetin as on ethyl butyrate, and the accelerations which WC have noted in experiments with triacetin are corre- spondingly less than with ethyl butyrato. In Series 4, where the activity on triacetin was less than usuaf the acceleration noted was greater. Experiments 8 of Series 4, IO and XI of Series 5, and IO to x4 of Series 6, show that 0.1 per cent. bile salts do not accelerate the action of the juice on olive oil, but when present in a concentrat~vn of 0.8 to 4 per cent, a very great acceleration is noted. The same is true of bile. When the biIe is so diluted that the bile salts are acting in a concentration of 0.1 percent. practically CO acceleration is noted (Esp. x I, Srrics 6).
On comparing the experimerIts in which bile salts and bile were used in similar concentrations, it will he seen that in many in- stances bile causes a greater acceleration than the bile salts. This is by no means invariably found to be the case as will be subse- quently shown, but these experiments taken alone would naturaljy lead to the conclusion th at both the bile salts and the lecithin or at Ieast some other constituent of the bile may play a part in the ac- celerating action. Several series of experiments were performed in order to see whether mixtures of the bile salts and lecithin cause a much greater acceleration than these substances do when acting individually; in other words, to see whether they give a summation effect. We have carried on these experiments with a series of esters in order to see whether the hydrolysis of the different esters is affected to the same degree by these substances,
SERIES 7. Duration of experiments, 18 hours. Temperature, 39O C. One C.C. of pancreatic juice was used in each experiment, It was diluted to 5 C.C. in every case. The bile salts acted in a concentration of 0.1 per cent., the lecithin in a 0.4 per cent. suspension; the mixture which will be designated “bile salts -I- lecithin” contained 0.1 per cent. of the former and 0.4 per cent. of the latter, and the experiments designated “bile” contained 0.1 C.C. of bile from the same dog which, on the supposi- tion that the bile contained 5 per cent. of bile salts would make the bile salts present in this experiment in a concentration of 0.1 per cent. as the total volume was 5 C.C. The figures as usual designate the number of cubic centimeters of & alkali required to neutralize the acid formed.
TABLP, A.
~~i~~~~~
---_---__ --------
The accelerations here noted are as follows: TABLE B .-PERCENTAGE ACCELERATIONS.
_ ____--_-.--_-.-- ..-- - .._. --__- ___..__ ----
Ester. Bile Salts. Lecithin. Bile Salts +
Lecithin. Bile.
Ethyl acetate 175 057 618 Ethyl ropionate
f3 3% 120
~
314 g;b&,utyrate 187 158 312 EX
119 Triacetin 58 2 ‘rli Ei
-___-- _-_-.--
I f we assign the value 100 to the hydrolysis of ethyl butyrate and thus determine the hydrolysis of the other esters in this series in terms of ethyl butyrate we have:
Table I? of this series shows that with this specimen of juice the percentage acceleration was much greater with the bile salts than with lecithin. The combined effect of lecithin and bile salts was less than that noted with the bile salts alone except in the case of the ethyl butyrate. Here the effect of both (312 per cent. acceleration) was nearly equal to the sum of their effect when acting separately, i. e. 345 per cent. Furthermore, it will be noted that the percentage acceleration caused by the bile salts alone in almost every case closely approximates that of the bile and leads to the conclusion that in this series of ex- periments, at least, the accelerating action of the bile is almost entirely dependent on the content of bile salts. The bile salts used were obtained from ox bile, which contains both tauro- chelates and glycocholates, and since they have an activity equal to that of dog bile in which only taurocholates are present, we would conclude that the effects of taurocholates and glyco- chelates are about equal. It is very interesting to note in these tables (especially C) the difference between the amounts of hydrolysis which t.he different esters suffered. Thus when no accelerator was used, the hydrolysis of the acetate was only 58 per cent. of that of the butyrate, while in the presence of the bile salts the hydrolysis of the acetate was 1.7 titnes that of the butyrate. Thus the effect of a given substance on the hydroly- sis of one ester is no index to its effect on the hydrolysis of an- other ester. We wish to emphasize one point especially and that is that the results vary with different experimental condi- tions, such as the dilution and quantity of the juice acting, the quantity of ester used, the temperature and duration of the experiments, and finally a considerable degree of variation is seen with different specimens of juice even when the experiments are duplicated as nearly as possible. In some cases lecithin causes a greater acceleration than the bile salts. This was usually found when the juice was employed in greater dilution.
A series similar to the above was carried through with olive oil.
SERIES 8. Duration of experiments, 19 hours. Temperature, 28* C, One-half of a cubic centimeter of pancreatic juice was used in each experi- ment. It acted in a total volume of 5 cc. in every case. The bile salts acted in a concentration of 2.6 per cent. both when used alone and with the lecithin. The lecithin again acted in a 0.4 per cent. suspension.
The bile was so diluted that the bile salts contained in it also acted in a concentration of 2.5 per cent. Six-tenths of a gram of olive oil was used in each experiment. The figures record the number of cubic centimeters of .“a alcoholic KOH required to neutralize the acid produced.
Water 10.85 11.22 Bile salts 19.25 18.75 67 Lecithin 12.3 12.4 11 Bile salts + lecithin 19.4 19.56 Bile - 17.9 - ii
_ _. -- ----_-,
Here again we see that the bile salts in the proper concentra- tion accelerate the action more than lecithin does and that the combined effect of lecithin and bile salts is very little greater than that of the latter when acting alone. Hammarstenl found the lecithin content of dog’s bile as discharged from a fistula to be about 0.12 per cent., hence in our experiments probably more lecithin was present per cubic centimeter of pancreatic juice than there is normally in the intestine. We have studied the action of pancreatic juice on lecithin under the conditions of our experi- ments. The increase in acidity noted was negligible and was not appreciably increased when bile salts were present. Schumoff- Simanowski and Sieber* have recently found that pancreatic juice actively hydrolyzes lecithin. In their experiments much larger quantities of lecithin were employed than in our work. In our work the increase in acidity due to the hydrolysis of lecithin was certainly not sufficiently great to materially vitiate the results.
COIWLUSIONS.
(I) Bile salts, lecithin, and bile greatly accelerate the action of pancreatic juice on all of the esters studied including o?ive oil.
(2) The optimum concentration for the bile salts when the
1 Jahresber. aber d. Fortsch. d. Thierchem., xxiii, p. 331, 1893. IIoppe- Seyler, L.ehrbwh der physiol. Chew, Berlin, 1881, p. 302.
* Zeitschr. f. pltysiol. Chem., xlix, p. 50, Igod.
loweresters are used is about 0.1 per cent., while for olive oil the the optimum is from 2 to 4 per cent. In the latter concentration the bile salts greatly inhibit the action on triacetin, and the ac- celeration of the hydrolysis of ethyl butyrate is much less than when they are employed in greater dilution.
(3) The combined effect of the bile salts and lecithin on the hydrolysis of the esters is usually but very little greater than that of the bile salts alone. Some notable exceptions to this were observed, however. Thus in some cases the acceleration was much greater when both were used, while in other casts the acceleration was quite a little less than when the bile salts were used alone.
(4) The effect of these accelerators on the hydrolysis of one ester is no index to the degree of acceleration which will be noted when other esters are used. One cannot predict what the acceleration will be even in an homologous series of esters.
(5) Different specimens of juice and different experimental conditions altered greatly the degree of acceleration observed and the relative activity of the bile salts and lecithin.
(G) The effects of bile salts and bile of a corresponding concentration of bile salts often very closely approximate one another, and we believe the bile salts are mainly responsible for the accelerating effect of bile on the hydrolysis of fats and esters by pancreatic juice.
(7) We advance no theory to account for the acceleration noted with these substances. While we believe that their action depends to a certain extent on their solvent action, it seems that in addition to this they accelerate the action of the enzyme in some other way.
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