THE QUESTION OF THE STABILITY OF SECRETIN
A. J. Carlson, A. E. Kanter and I. Tumpowski
[From the Hull Physiological Laboratory of the University of Chicago]
In a letters patent, filed May 6, 1914, the patent granted May 2, 1916, James W. Beveridge, M.D., makes certain claims concerning the stability and physiologic activity of secretin prepared according to the method patented by him.
In brief, Dr. Beveridge claims that secretin prepared by digesting intestinal mucosa with a weak acid at a temperature slightly below boiling, and mixed with 0.2 per cent. to 2 per cent. blood serum, albumin or peptone (1) remains active for at least six months, (2) stimulates the pancreas when given by mouth, and (3) “may be injected intravenously in man, if desired.” The only thing in the letters patent in support of these claims is the statement: “I have found out by actual tests that the preparation maintains its stability for five or six months.”
Here are the claims in detail:
“For the source of secretin I preferably use that part of the alimentary tract of any lower animal—such as a hog or sheep—including the gastric pylorus, the duodenum and the jejunum. This part is split open and washed with a normal saline solution to clean the mucosa or mucous membrane of any detritus which may be present. The mucosa with the epithelial cells is then removed or separated from the muscular wall by scraping with a blunt knife or in any other suitable way. The scrapings or cuttings, which contain the secretin, are then macerated or broken up.”
“The macerated mass is placed in a suitable vessel and subjected to the action of an acid solution until digested. The time for the digestion of the mass will, of course, depend upon the strength and temperature of the acid solution employed. The stronger the solution and the higher the temperature, the shorter the time necessary for complete digestion. This period may vary from several minutes to several hours. In my experiments I found that the best results were obtained with hydrochloric acid solution of one-tenth to five-tenths of one per cent. in strength, although as high as eight-tenths per cent. might be used. The mixture is brought to a temperature of approximately 210 F., and it may even for a few moments exceed that temperature, but it should be kept below the boiling point, for excessive heat injures or breaks down the secretin molecule and impairs or destroys its activity. Although I prefer to use hydrochloric acid, I would have it understood that other acids—both organic or inorganic—may be employed, provided that the percentage of acidity is regulated to prevent a chemical change in the secretin, and further provided, of course, that the acid has no injurious effect on the human system.”
“After the mass has been digested in the heated solution, the decoction is decanted, and after being allowed to cool is passed through a suitable filter until the filtrate is clear. I found that by filtering the decoction from four to six times through a carbon filter, I obtained a clear colorless filtrate. This is a solution of secretin and the acid which was used, and the clearness of the solution shows that it is practically free from albumoses, gelatin and other impurities (such as cell tissues, etc.) present in the raw material under treatment.”
“To the solution of pure and active secretin prepared as above explained, there is added a suitable quantity of blood serum—say from one-fifth to two per cent. or any equivalent medium—such as albumin solution or a peptone solution—which will aid and sustain the activating power of secretin as provided by the blood. That is to say, any medium having the same power, similar quality or chemical composition that the blood-stream possesses in combining with secretin to stimulate the pancreas. The addition of such a medium to the active secretin solution increases the potency of the secretin and its degree of stability by preventing oxidation or deterioration thereof. If this strengthening or fortifying medium, as it may be properly termed, is alkaline, it performs the additional function of lowering the acidity of the secretin filtrate. It is preferable that the final product be just faintly acid. If desired, the final product may be made into an elixir by the addition of aromatics.”
“Any desired strength of secretin solution may be obtained according to the quantity of acid solution. In my experiments I used from ten to fourteen duodena to a pint of acid solution.”
“The solution of secretin prepared as above described is characterized by its ability to resist oxidation or deterioration for a sufficient period of time to render the solution available as a commercial article, and is furthermore characterized by freedom from poisonous and irritable chemical substances, whereby the secretin is chemically adapted to the human system to stimulate the pancreas to increased secretion.”
“As previously stated, the secretin prepared according to my method may be administered orally to produce the desired physiological action. Of course, if desired, the secretin might be injected intravenously, but this more or less dangerous procedure is not at all necessary, and I merely mention it here to point out that when I refer to the oral administration of my new secretin preparation, I do not mean to exclude its administration by injection.”
“As to the commercial stability of the secretin prepared according to my method, I may say that I have found by actual tests that the preparation maintains its stability for as long a period as five or six months. When I refer to my product as being “commercially stable,” I mean that it resists oxidation or deterioration for a sufficient period to render the same available as a commercial article. This period may vary from several weeks to several months, depending upon certain commercial factors well understood by the manufacturer. So, roughly speaking, I should say that secretin is commercially stable when it retains its activity from one to six months. I do not wish to be understood, however, as limiting myself to these exact figures.”
That active secretin may be extracted from macerated intestinal mucosa by weak acids below the temperature of boiling is well known. In fact, weak acids at body temperature in contact with the duodenal mucosa lead to the formation of secretin. The claims that secretin given by mouth reaches the blood and acts on the pancreas has been made for other preparations of secretin. It has also been shown that these claims are erroneous.[122] Thus it would appear that the only novel element in Dr. Beveridge’s patented secretin is the addition of serum, soluble proteins or peptones. What reason is there for believing that this will render the secretin stable for months, and physiologically active when taken by mouth? We do not believe Dr. Beveridge ever injected his secretin—protein mixture—intravenously in man or animals not under anesthesia, otherwise he would not have stated: “Of course, if desired, the secretin may be injected intravenously.”
BEVERIDGE’S PATENTED SECRETIN IS NOT STABLE
I. The Samples of Secretin Sent Us by Dr. Beveridge.—Physiological tests were made on four quart bottles of the secretin kindly sent us by Dr. Beveridge June 26, 1916. According to a letter from Dr. Beveridge of July 20, 1916, those samples of secretin were prepared June 20, that is, only six days before received by us. The material came in dark colored bottles. It was kept in the original bottles and placed in the ice box immediately on receipt. Dr. Beveridge stated the secretin “should remain active until the month of November, 1916, at least.”
Tests were made on three out of the four bottles. The fourth bottle was not opened, as we desired to learn what change it might undergo in the way of protein precipitation and bacterial decomposition. There is nothing in the Beveridge method of preparation that insures a sterile secretin unless it is passed through a Berkefeld filter. In all our crucial experiments the animals (dogs) were kept under light ether anesthesia, a cannula inserted into the pancreatic duct, the blood pressure recorded from the carotid artery and the various secretin preparations injected intravenously. When inactive secretin preparations were encountered, control tests were always made with active solutions of secretin to eliminate possible individual peculiarities of the animal. Thus when the pancreas of a dog reacts to the injection of preparation A, but not to preparation B, it is evident that absence of response to B is due to this preparation and not to the animal or to the experimental conditions.
Fig. 1.—Records of carotid blood pressure and secretion of pancreatic juice on intravenous injection of Beveridge’s secretin in dogs. X, injection of 10 c.c. secretin; b, record of flow of pancreatic juice in drops. Tracing A, injection of 10 c.c. of one sample secretin (ten days old) furnished by Dr. Beveridge. Tracing B, injection of 10 c.c. of second sample of secretin (ten days old) furnished by Dr. Beveridge. Tracing C, injection of 10 c.c. of secretin (twenty hours old) made by us according to the Beveridge method. Showing that the secretin preparations sent us by Dr. Beveridge contained no secretin.
Each of the three samples of secretin sent us by Dr. Beveridge was tested in the above manner on five dogs. The first tests were made June 27, 28 and 29, respectively, that is, within nine days of the preparation of these samples of secretin. None of the samples was active (Fig. 1), even when injected intravenously in quantities up to 50 c.c.: 40–50 c.c. of Beveridge’s secretin mixture may kill a dog by too great lowering of the blood pressure. A good secretin preparation yields a copious secretion of pancreatic juice on intravenous injection of a few cubic centimeters.
It is not difficult to prepare a secretin, by the original Bayliss or Starling method or by the Beveridge method, that retains some activity for a longer period than nine days. Hence we cannot account for the absolute inactivity of these preparations except on the assumption that they did not contain any secretin to start with; that is, faulty preparation and absence of physiologic standardization.
The sample kept intact in its original container for six months became gradually cloudy, a large mass of amorphous precipitate settled to the bottom and the odor showed bacterial decomposition. It is reprehensible, to say the least, to state concerning such a mixture: “Of course, if desired, it may be injected intravenously.” The fact that Beveridge’s secretin may be rendered clear by filtering through carbon is not sufficient evidence that it is “pure secretin,” free from bacteria and other injurious substances.
II. Beveridge Secretin Mixture Is Rapidly Rendered Inactive by Human Gastric Juice.—We prepared active secretin solutions by the Beveridge method, using 0.2 per cent. serum as the protein “stabilizer” (?). The addition of the serum does not appear to affect the activity of the fresh secretin preparation. If Beveridge’s secretin is able to act on the pancreas when given by mouth, it is obvious that it must run the gamut of gastric digestion, except in cases of complete achlorhydria. It has been repeatedly demonstrated that all other secretin preparations are rapidly destroyed by pepsin-hydrochloric acid digestion. Is Beveridge’s secretin an exception? What is there in a little serum, native albumin, or peptones to protect secretin against gastric digestion?
The pure human gastric juice used in these tests was secured from the fistula case (Mr. F. V.) that has been under observation in our laboratory for years.[123]
BEVERIDGE’S SECRETIN AND BAYLISS-STARLING SECRETIN PREPARED
Sept. 29, 1916
| Date of Test | Quantity of Secretin Injected, C.c. | Response of Pancreas (No. of Drops of Secretin) | |
 | |||
| Bayliss-Starling Secretin | Beveridge Secretin | ||
Sept. 29 | 10 | 75 | 78 |
Oct. 2 | 10 | 61 | 61 |
Oct. 6 | 10 | 28 | 17 |
Oct. 13 | 10 | 25 | 31 |
Oct. 27 | 10 | 5 | 6 |
Nov. 3 | 10 | 7 | 6 |
Nov. 17 | 10 | 4 | 5 |
Nov. 30 | 10 | 3 | 4 |
Dec. 4 | 10 | 2 | 2 |
Dec. 20 | 10 | 0 | 0 |
Two cubic centimeters of fresh gastric juice added to 8–10 c.c. Beveridge secretin, the mixture being kept at body temperature (38 C.), renders the secretin completely inactive in from 5 to 8 minutes (Fig. 2). There is no exception to this rule, as we have repeated the test on many different secretin preparations and using different samples of human gastric juice. The secretin of Beveridge is just as vulnerable as the secretin of Bayliss and Starling to pepsin-hydrochloric acid digestion. On what kind of tests does Beveridge base his claim that his secretin mixture acts on the pancreas when given by mouth?
III. The Relative Rate of Deterioration of the Secretin Solutions Prepared According to Bayliss and Starling and According to Beveridge.—Six different preparations of the two kinds of secretin were made, kept in dark stoppered bottles in the ice box, and tested by intravenous injection in dogs under ether anesthesia from time to time until all influence on the pancreas had been lost. One typical series of these tests is given by the way of illustration. (See Table on page [126].)
Fig. 2.—Records of carotid blood pressure and flow of pancreatic juice on intravenous injection of secretin prepared by us according to the Beveridge method. X, injection of 10 c.c. of the secretin; b, record of flow of pancreatic juice in drops. Tracing A, the 10 c.c. of Beveridge’s secretin injected had been digested for five minutes with 3 c.c. of human gastric juice. Tracing B, injection of 10 c.c. of the same secretin preparation not subjected to gastric digestion. Showing rapid and complete destruction of Beveridge’s secretin by human gastric juice.
It will be seen that the rate of deterioration (oxidation or decomposition) of the secretin is practically the same whether prepared according to Bayliss and Starling or according to Beveridge (Figure 3). In both preparations the rate of deterioration is most rapid the first few days after preparation. It is scarcely necessary to point out that secretin preparations not kept constantly at low temperature and in the dark, as in the above experiments, will deteriorate more rapidly.
Fig. 3.—Records of carotid blood pressure and flow of pancreatic juice on intravenous injection of secretin preparations. X, injection of 10 c.c. secretin; b, record of flow of pancreatic juice in drops. Tracing A, secretin prepared according to the Beveridge method September 30. I, injection of 10 c.c. October 2. II, injection of 10 c.c. November 30. Tracing B, secretin prepared by the Bayliss-Starling method September 30. III, injection of 10 c.c. October 2; IV, injection of 10 c.c. November 30. Showing no greater stability of Beveridge’s secretion over that of Bayliss and Starling.
Why can we hope that the addition of serum or any solution of protein will render secretin more stable? In the intact man or animal under normal conditions of digestion, secretin reaches the pancreas by way of the blood, that is, it is in solution in blood. Does that fact render the secretin stable? By no means. The reader is familiar with the fact that the response of the pancreas to a single intravenous administration of secretin is very transitory (5–15 min.). The cessation of activity is due, not to fatigue of the pancreas, as a second injection of secretin gives a prompt response of pancreatic secretion, but to the disappearance of active secretin from the blood. In fact, secretin left in the test tube or in the bottle remains active over a much longer period of time than when introduced into the blood stream.
IV. Beveridge’s Secretin Given by Mouth to the Intact Animal Has No Specific Action on the Pancreas.—Active secretin prepared according to the method of Beveridge was fed on an empty stomach to a small dog (5 kilo) with permanent fistula of one of the pancreatic ducts. On control days we gave the dog (a) equal quantities of n/10 HCl, and (b) bread and milk. The Beveridge secretin was prepared with 0.3 per cent. HCl and the addition of 0.2 per cent. serum. The results may be stated by the following summary:
GIVING BEVERIDGE’S SECRETIN BY MOUTH
| Material Fed | Number of Tests | Secretin of the Pancreas for Three Hours Following the Feeding |
| 150 c.c. Beveridge Secretin | 6 | 10.2 c.c. |
| 150 c.c. n/10 HCl | 5 | 22.7 c.c. |
| Bread soaked in milk | 4 | 6.6 c.c. |
The Control experiments with pure hydrochloric acid show that the secretion of pancreatic juice following the introduction of Beveridge’s secretin into the stomach is due to the acid factor and the protein content.
CONCLUSIONS
The patented secretin of Beveridge is rendered inactive by gastric juice, is without effect when given by mouth, and exhibits no greater stability or keeping qualities than the secretin prepared according to Bayliss and Starling. It has no merit as a therapeutic agent. It should under no conditions be administered intravenously in man, as it contains deleterious protein split products and living bacteria.—(From The Journal A. M. A., Jan. 12, 1918.)