Native Proteid.Syntonin.Protoproteose.Heteroproteose.(dysproteose).Deuteroproteose.Deuteroproteose.Peptone.Peptone.
It is, of course, to be understood that this is not intended to represent anything more than the order of formation of the several bodies, no attention being paid here to the hemi- or anti-character of the several products, or classes of products. Thus, proto and heteroproteose are primary bodies formed directly from the initial product syntonin by the further action of the ferment. In the same sense, deuteroproteose is a secondary proteose, being formed by the further hydration of the primary body. Lastly, peptones, the final products of pepsin-proteolysis, are the result of the hydration and possible cleavage of deuteroproteoses. Further, in almost every gastric digestion there is also formed a small amount of antialbumid, a product insoluble in dilute hydrochloric acid and which consequently appears as an insoluble residue. This body is very resistant to the action of pepsin-acid when once formed, but may be slowly converted, in part at least, into a soluble antialbumose and thence into antipeptone.
All of these bodies can be readily identified in any digestive mixture containing them by a few simple reactions. Thus, after having removed any acid-albumin or syntonin present by neutralization, the concentrated fluid can be tested at once. If primary proteoses are present, the neutral fluid will yield a more or less heavy precipitate on addition of crystals of rock-salt, precipitation being complete only when the fluid is saturated with the salt. Further, if the proteoses are present in not too small quantity, nitric acid added drop by drop to the neutralized fluid will produce a white precipitate, readily soluble on application of heat but reappearing as the solution cools. If primary proteoses are wholly wanting, then no precipitate will be obtained by acid unless the fluid is saturated with salt, in which case a portion of the deuteroproteose will be precipitated. The two primary proteoses differ from eachother especially in solubility; protoproteose being readily soluble in water alone, while heteroproteose is soluble only in salt solutions, dilute acids, and alkalies. Hence, when these two bodies are precipitated together by saturation with salt, they may be readily separated by dissolving them in a little dilute salt solution, and dialyzing the fluid in running water until the salt is entirely removed; heteroproteose will then be precipitated, while the proto-body remains in solution.
By long contact with water, and even with concentrated salt solutions, heteroproteose tends to undergo change into a semi-coagulated form, named dysproteose, insoluble in dilute sodium-chloride solutions. This body can be reconverted into heteroproteose, in part at least, by solution in dilute acid, or alkali, and reprecipitation by neutralization.
As a class, the proteoses are characterized by far readier solubility in water than native proteids, by a far greater degree of diffusibility, by non-coagulability by heat and by alcohol, although precipitable by the latter agent. Further, nearly all proteose precipitates are exceedingly sensitive toward heat, tending to dissolve as the fluid is warmed and reappearing as the solution cools. In fact, this peculiarity often serves as a means of identification. Potassium ferrocyanide and acetic acid, picric acid in excess, and likewise cupric sulphate, all precipitate the primary proteoses, while deuteroproteose is only slightly affected by these reagents, or indeed not at all.
In order to separate the secondary proteose from the primary bodies in the absence of peptones, the fluid is neutralized as nearly as possible, and then, after suitable concentration, is saturated with sodium chloride for the partial precipitation of the primary proteoses. To the clear filtrate, aceticacid117is added drop by drop as long as a precipitateresults, the latter being composed of a mixture of protoproteose and deuteroproteose. That is to say, protoproteoses are not completely precipitated from neutral solutions by saturation with salt alone; a little acid is required to complete it, but this tends to bring down a certain amount of deuteroproteose. From this filtrate, however, the deutero-body can be separated in a pure form by dialyzing away the salt and acid, and then concentrating the fluid and precipitating with alcohol. When the proteoses are mixed with peptones, the former must first be separated collectively by saturation of the fluid with ammonium sulphate.
Peptones are especially characterized by non-precipitation with the ordinary precipitants for proteid bodies, and especially by the fact that they are wholly indifferent to saturation with ammonium sulphate either in neutral, acid, or alkaline fluids. This reaction, which constitutes the main, and perhaps the only absolute method of separating peptones from proteoses must be carried out with great thoroughness in order to insure a complete precipitation of deuteroproteose. The latter stands midway between primary proteoses and peptones in many respects, and seems to share with peptones something of a tendency to resist precipitation by the ammonium salt. Indeed, asKühne118has recently pointed out, the last traces of deuteroproteose can be precipitated from the fluid only by long continued boiling of the ammonium sulphate-saturated fluid, and even then it is seldom complete unless the reaction of the fluid is alternately made neutral, acid, and alkaline, and the heating continued for some time after each change in reaction. Under such circumstances, the last portions of deuteroproteose separate from the salt-saturated fluid and float on the surface in the form of an oily or gummy mass, while thetrue peptone remains in the fluid absolutely non-precipitable by the salt.
In this filtrate, peptone can be detected by adding to a small portion of the fluid a very large excess of a strong solution of potassium hydroxide, followed by the addition of a few drops of a very dilute solution of cupric sulphate. If peptone is present a bright red color will appear, the intensity of which, with the proper amount of cupric sulphate, will be proportional to the amount of peptone present. If it is desired to separate the peptone from the ammonium-sulphate-saturated fluid, there are several methods available, of which the following is perhaps the most satisfactory: The fluid is concentrated somewhat, and set aside in a cool place for crystallization of a portion of the ammonium salt. The fluid is then mixed with about one-fifth its volume of alcohol, and allowed to stand for some time, when it separates into two layers—an upper one, rich in alcohol, and a lower one, rich in salts. The latter is again treated with alcohol, by which another separation of the same order is accomplished. Finally, the lighter alcoholic layers containing the peptone are united, and exposed to a low temperature until considerable of the contained salt crystallizes out. The fluid is then concentrated, and after addition of a little water is boiled with barium carbonate until the fluid is entirely free from ammonium sulphate. Any excess of baryta in the filtrate is removed by cautious addition of dilute sulphuric acid, after which the concentrated fluid, reduced almost to a sirupy mass, is poured into absolute alcohol for precipitation of the peptone.
So separated, the peptone formed in gastric digestion is exceedingly gummy, but can be transformed into a yellowish powder, very hygroscopic, of more or less bitter taste, and, when thoroughly dry, dissolving in water with a hissing sound and with considerable development of heat, like phosphoric anhydride.119
I have introduced these dry chemical facts, none of which are especially new, because I deem them of considerable importance and because they are not very generally known. In fact, there seems to be a tendency on the part of some who are more or less familiar with the advances made in our knowledge of the products of pepsin-proteolysis to question the existence of these different bodies, or to show at least a spirit of indifference toward these recent facts which have been gradually accumulated, and I may say accumulated at the expense of considerable labor. The time is past for calling the products of gastric digestion peptones; it is time for a full recognition of the fact that pepsin-proteolysis is synonymous with the production of a row of bodies, chemically and physiologically distinct from each other, each endowed with individuality enough to admit of certain detection, and all bearing a certain specific and harmonious relationship to their neighbors, the other members of the series.
Further, it is not enough to admit the formation of a single intermediate body, midway between syntonin and peptone. The so-called propeptone of the past is simply a mixture of proteoses, of ever changing composition, varying with each change in the proportion of the component proteoses. Each of these proteoses can be detected, under suitable conditions, in the products of every artificial digestion as well as in the stomach-contents, and no better measure of the proteolytic power of the natural stomach-secretion can be devised than a study of the character of the individual bodies present in the stomach-contents after a suitable test meal. The proper tests and separations canbe made with a small amount of the filtered fluid, and much light thrown upon the digestive power of the secretion by even a rough estimate of the proportion of primary and secondary proteoses and peptones formed in a given time, after the ingestion of a certain amount of proteid food.
In pepsin-proteolysis we have to deal, in my opinion, with a series of progressive hydrolytic changes in which peptones are the final products of the transformation. Commencing with the formation of acid-albumin or syntonin, hydrolysis and cleavage proceed hand in hand, under the guiding influence of the proteolytic enzyme, and each onward step in the process is marked by the appearance of a new body corresponding to the extent of the hydrolysis; each body, perhaps, being represented by a row or series of isomers, all externally alike, but different in their inner structure, according to the proportion of hemi- and anti-groups contained in the molecule. As opposed to this theory, we have the older views of Maly,120Herth,121Henninger122and others, based upon observations which tend to show that peptones do not differ in chemical composition from the proteids which yield them. As a matter of fact, the products then analyzed were not peptones at all; they were merely theprimaryproducts of pepsin-proteolysis,i. e., what we now term primary proteoses, and it is time we stopped using such data to enforce the theory that peptones are polymers of the proteids from which they are derived.
In 1886, the writer, in conjunction with Professor Kühne, commenced a study of the various cleavageproducts123formed by the action of pepsin-hydrochloric acidfrom the better characterized and purer proteids, this being a continuation of our earlier work on the proteoses and peptones formed from blood-fibrin, serum-albumin, etc. This work I have continued in my laboratory up to the present time, with many co-workers, and as a result we have to-day a series of observations gradually accumulated during these last seven years, some the results of work carried on this last year, which speak in no uncertain way of the character of both the primary and secondary products of pepsin-proteolysis. Furthermore, in attempting to settle this question once for all, I have selected for study examples from the various classes of both animal and vegetable proteids; and as representatives of the latter have had carried out two lengthy series of experiments on the crystallized proteids which occur so abundantly in some seeds, on the assumption that these crystalline bodies would furnish a certain guarantee of purity which might naturally be lacking in the amorphous proteids of animal origin. Some of these results are now placed together in the following tables, a study of which reveals some very interesting facts:
COMPOSITION OF PROTEOLYTIC PRODUCTS FORMED BY PEPSIN-HYDROCHLORIC ACID.
Proteolysis of Blood-fibrin.
Mother Proteid.Protofibrinose.124Heterofibrinose.124Deuterofibrinose.124Amphopeptone.125C52.6851.5050.7450.4748.75H6.836.806.726.817.21N16.9117.1317.1417.2016.26S1.100.941.160.870.77O22.4823.6324.2424.6527.01
Proteolysis of Paraglobulin.126
Mother Proteid.Protoglobulose.Heteroglobulose.Deuteroglobulose.C52.7151.5752.1051.52H7.016.986.986.95N15.8516.0916.0815.94S1.1125.3624.8425.59O23.24
Proteolysis of Coagulated Egg-albumin.
Mother Proteid.Protoalbumose.127Heteroalbumose.127Deuteroalbumose.127Hemipeptone.128C52.3351.4452.0651.1949.38H6.987.106.956.946.81N15.8416.1815.5515.7715.07S1.812.001.632.021.10O23.0423.2823.8124.0827.64
Proteolysis of Casein from Milk.
Mother Proteid.Protocaseose.129Heterocaseose.130α Deuterocaseose.129β Deuterocaseose.129C53.3054.5853.8852.1047.72H7.077.107.276.936.73N15.9115.8015.6715.5115.97S0.8222.5223.1825.4629.58O22.03
Proteolysis of Myosin from Muscle.131
Mother Proteid.Protomyosinose.Deuteromyosinose.C52.8252.4350.97H7.117.177.42N16.7716.9217.00S1.271.321.22O21.9022.1623.39
Proteolysis of Elastin.132
Mother Proteid.Protoelastose.Deuteroelastose.C54.2454.5253.11H7.277.017.08N16.7016.9616.85S21.7921.5122.96O
Proteolysis of Gelatin.133
Mother Proteid.Protogelatose.Deuterogelatose.C49.3849.9849.23H6.816.786.84N17.9717.8617.40S0.710.520.51O25.1324.8626.02
Proteolysis of Phytovitellin134(Crystallized) from Squash Seed.
Mother Proteid.Protovitellose.Deuterovitellose.C51.6051.5249.27H6.976.986.70N18.8018.6718.78S1.0122.8325.25O21.62
Proteolysis of Phytovitellin135(Crystallized) from Hemp Seed.
Mother Proteid.Protovitellose.Deuterovitellose.Peptone.C51.6351.5549.7849.40H6.906.736.736.77N18.7818.9017.9718.40S0.901.091.080.49O21.7921.7324.4424.94
Proteolysis of Glutenin136from Wheat.
Mother Proteid.Protoglutenose.Heteroglutenose.Deuteroglutenose.C52.3451.4251.8249.85H6.836.706.796.69N17.4917.5617.4317.57S1.081.341.590.80O22.2622.9822.3725.09
Proteolysis of Zein.137
Mother Proteid.Protozeose.Deuterozeose.C55.2353.2951.31H7.266.876.88N16.1316.1016.27S0.601.541.08O20.7822.2024.46
In considering these results, it is to be noticed that there is a general unanimity of agreement except in the case of the albuminoid gelatin. In the proteolysis of this body, for some reason not explainable, the digestive products show no marked deviation from the composition of the mother-proteid, but in every other instance there is to be traced a distinct tendency toward diminution in the content of carbon, proportional to the extent of proteolysis. In the primary bodies, proto and heteroproteoses, the percentage of carbon is only slightly lowered; indeed, in some few cases, notably in elastin and casein, the primary products show a slight increase in their content of carbon, but in most instances there is a slight falling off in the percentage of this element. In the deuteroproteoses, however, the loss of carbon is very marked. The percentage loss, to be sure, varies with the different proteids, doubtlessdependent in part upon the nature of the proteid itself, and also, I think, upon the strength of the proteolytic agent employed and the duration of the proteolysis. It is to be further noticed that peptones, whenever analyzed, show a still further loss of carbon and also a marked loss of sulphur. In nitrogen there is no constant difference.
On the assumption that these various products of proteolysis are formed by a series of hydrolytic changes, accompanied by cleavage of the molecule, we might at first glance look for a marked increase in the content of hydrogen. But when we consider the size of the proteid molecule, with the small proportion of hydrogen contained therein and the large amount of carbon, it is plain that hydrolytic cleavage might naturally leave its mark on the percentage of carbon, rather than on the percentage of hydrogen of the resultant products. In view of these facts, the above results show nothing inconsistent with the theory that pepsin-proteolysis, as a rule, is accompanied by a series of progressive hydrolytic cleavages in which the primary proteoses are the result of a slight hydration, these bodies by continued proteolysis being further hydrated with formation of secondary proteoses, which in turn undergo final hydration and cleavage into true peptones. In accord with this theory, true peptones always show a marked difference in composition from that of the mother-proteid, the most striking feature being the greatly diminished content of carbon, which may be taken as a measure, in part at least, of the extent of the hydrolytic change. And it is to be noticed that the crystallized phytovitellins are no exception to the general rule; the secondary vitelloses and peptones resulting from proteolysis bear essentially the same relationship to the mother-proteids that the albumoses from egg-albumin do. Moreover, the alcohol-soluble proteids, of which the zein of cornmeal is a good example,show the same general tendency, and it is an interesting fact that the proteoses, or more specifically the zeoses, formed from this peculiar proteid, are readily soluble in water and show the general proteose reactions. It may also be mentioned that these zeoses, as well as the elastoses, are very resistant to further hydrolysis by pepsin-acid, and yield only comparatively small amounts of true peptones.
In connection with this question of the composition of proteoses and peptones as formed by pepsin-proteolysis, it is interesting to note a recent observation recorded by Schützenberger.138This experimenter took 350 grammes of moist blood-fibrin, corresponding to 75.5 grammes of dry substance, and subjected it to proteolysis with 2.5 litres of a very strong pepsin-hydrochloric acid solution for five days. The resultant fluid was then freed from acid by treatment with silver oxide, after which the solution was evaporated to dryness on a water-bath and the residue driedin vacuo. This residue, termed by Schützenberger fibrin-peptone, was found on analysis to contain 49.18 per cent. of carbon, 7.09 per cent. of hydrogen, and 16.33 per cent. of nitrogen, thus agreeing very closely with true fibrin-peptone as analyzed by Kühne and myself. Further, Schützenberger showed that the fibrin in undergoing this transformation had taken on 3.97 per cent. of water. But to my mind, the most significant fact connected with this experiment is the positive evidence it affords, not only of hydration as a feature of peptonization by pepsin-acid, but that this greatly diminished content of carbon, so characteristic of peptones, and to a less extent of deuteroproteoses, is wholly independent of the methods of separation and purification ordinarily made use of. Thus, Schützenberger, in the above experiment, did not attempt any separation ofindividual bodies. Proteolysis was carried out under conditions favoring maximum conversion into peptone, and the resultant product, or products, was analyzed directly without recourse to any methods of precipitation or purification. To be sure, the substance analyzed could not have been peptone entirely free from proteose, but in any event it represented the terminal products of pepsin-proteolysis, and like true amphopeptone contained 3.5 per cent. less carbon than the original fibrin. Hence, we may conclude, without further argument, that peptonization in gastric digestion is the result of distinct hydrolytic action, in which the original proteid molecule is gradually broken down, or split apart, into a number of simpler molecules, the proteoses and peptones.
Peptones,i. e., amphopeptones, are the final products of gastric digestion; but to how great an extent is actual peptonization carried on in pepsin-proteolysis? As we have seen, syntonin, primary proteoses, secondary proteoses, and peptones are all products of pepsin-digestion, and it might perhaps be assumed that ultimately all of a given proteid undergoing pepsin-proteolysis would be converted into amphopeptone. Examination, however, shows that such is not the case, at least in artificial digestive experiments. Peptones are truly formed, and many times in large amount, but never under any circumstances have I been able to effect a complete transformation of any proteid into true peptone by pepsin-proteolysis; there is always found a certain amount of proteoses more or less resistant to the further action of the ferment. Obviously, the nature and proportion of the individual products formed in any digestive experiment are dependent greatly upon the attendant conditions; but even with a large amount of active ferment, an abundance of free hydrochloric acid, a proper temperature, and a long-continuedperiod of digestion, even five and six days, there is never found a complete conversion into peptone. Indeed, the largest yield of peptone I have ever obtained in an artificial digestion is sixty per cent., while the average of a large number of results under most favorable circumstances is somewhat less than fifty per cent.139
We understand that peptones are the products of the hydration and cleavage of previously formed proteoses. The primary proteoses pass into secondary proteoses and these into peptones, but for some reason this transformation after a time becomes a slow and gradual process. At first there is a marked and rapid progression; the proteid undergoing proteolysis is rapidly dissolved, and both proteoses and peptones may be detected in abundance. But if we continue to watch the changing relations of primary and secondary proteoses and peptones, we find that progression soon ceases to be rapid, and eventually travels onward at a snail’s pace. Thus, in one experiment with coagulated egg-albumin, there was found at the end of forty-eight hours’ digestion with pepsin-hydrochloric acid, only thirty-seven per cent. of peptones with fifty-eight per cent. of proteoses, and yet digestion had been sufficiently vigorous to allow of a complete solution of the proteid in two hours. At the end of seventy-two hours the amount of peptones had increased to about forty-two per cent., the proteoses having correspondingly diminished; but even at the end of seventeen days only fifty-four per cent. of peptones were to be found, thus affording striking evidence of the slow conversion of the first-formed products into peptones.
Naturally, the individual proteoses show marked differences in their rate of conversion into secondary or finalproducts. Take as an illustration someresults140obtained with caseoses formed in the digestion of the casein of milk. Thus, heterocaseose, a primary product, yielded only fifteen per cent. of peptone after ninety-four hours at 40° C. with a strong pepsin-acid solution. Protocaseose, however, containing some deuterocaseose, under like conditions, yielded thirty-two per cent. of peptone in one hundred and nineteen hours, while pure deuterocaseose gave sixty-six per cent. of peptone in one hundred and thirty-seven hours. Evidently, then, the first-formed soluble products of gastric digestion,i. e., the primary proteoses, are only slowly converted into peptone, since they must first pass through the intermediate stage of deuteroproteose, which is plainly not a rapid process. The deutero-body, on the other hand, once formed is more rapidly converted into peptone, but even this is in no sense a rapid process. Hence, in the artificial digestion of proteids with pepsin-hydrochloric acid, solubility of the proteids may be quite rapid, and even complete in a very short time, but the resultant products will be mainly proteoses and not peptones. The latter are truly formed and in considerable amount, but proteoses, either as primary or secondary bodies, are invariably present and usually in excess of the peptones.
In this connection the question naturally arises how far we are to trust these results in their bearing on the natural process of digestion as it occurs in the living stomach. Obviously, the conditions are quite different in the two cases. In artificial digestions, we have especially the influence of an ever-increasing percentage of soluble products on the activity of the ferment, a condition of things generally considered as more or less inhibitory to enzyme action. We have attempted to measure the real value ofthis influence byexperiments141conducted in parchment dialyzing tubes, in which the conditions are made favorable for the removal of at least some of the products of digestion as fast as they are formed. In these experiments, the dialyzer tubes containing the proteid and pepsin-acid were immersed in a large volume of 0.2 per cent. hydrochloric acid (about three litres), which was gradually changed from time to time, the whole mixture being kept at 40° C. during the entire period of the experiment. The extent of peptonization was then ascertained by analysis of both the contents of the dialyzer tubes and of the surrounding acid, the results being compared with those obtained from control experiments carried on in flasks. Without considering the results in detail, it may be mentioned that the slow and incomplete peptonization so characteristic of artificial gastric digestion is not materially modified by this closer approach to the natural process. The several digestions carried on in the dialyzer tubes were certainly accompanied by a fairly rapid withdrawal of the diffusible products of digestion, yet no noticeable increase in the amount of peptone formed was observed. The results certainly favor the view that the conversion of the primary products of gastric digestion into true peptone is a slow and gradual process, even under the most favorable circumstances, and that this lack of complete peptonization is not due to accumulation of the products of digestion, but is rather an inherent quality of pepsin-proteolysis under all circumstances.
In these dialyzer experiments it was observed that not only did peptones diffuse, but also the proteoses. In fact, it was found that six to eight per cent. of the proteosesformed passed through the parchment walls of the dialyzer tubes into the surrounding acid in the nine hours’ digestion. This led to a study of the diffusibility of proteoses in general, from which we were led to conclude that these bodies possess this power to a greater degree than had hitherto been supposed. As might be expected, it was also found that the attendant conditions modify materially the rate of diffusibility; the two factors especially prominent being temperature and the volume of the surrounding fluid. Thus, 1.9 grammes of protoalbumose dissolved in 200 c.c. of water and suspended in 4.5 litres of water heated to 38° C., diffused through the parchment tube to the-extent of 5.09 per cent., while at 10° C. diffusion amounted to only 2.57 per cent. Under somewhat similar conditions, pure peptone diffused to the extent of eleven per cent. in six hours at 38° C. Somewhat singular, however, was the result obtained with deuteroalbumose; this proteose showing a diffusibility considerably less than that of the proto-body. But asKühne142has independently obtained essentially the same results, this apparent anomaly cannot depend upon any errors of work.
It is of course to be understood that diffusion experiments made with dead parchment membranes cannot necessarily be expected to throw much light upon the rate of absorption of these bodies through the living membranes of the stomach and intestine, where, as WaymouthReid143has well said, we have to deal with an absorptive force dependent, no doubt, upon protoplasmic activity, and comparable, in part at least, to the excretive force of a gland-cell. Furthermore, in considering absorption as it occurs in the living stomach, we must necessarily give due weightto the selective power of the epithelial cells, a power which may be far more potent even than we suppose. Hence, without attempting at this point to draw any broad deductions from our experiments we may simply lay stress upon the facts themselves, viz., that the primary products of pepsin-proteolysis are diffusible, and, like true peptones, are capable of passing through animal and vegetable membranes, although to a less extent. We may further emphasize the fact that experiments of this character on diffusibility can, at the most, only indicate general tendencies, since every variation in the attendant conditions will exercise some influence upon the final result.
With reference to the bearing digestive experiments made in dialyzer tubes have upon the natural process as carried on in the living stomach, we must necessarily grant that the conditions approximate only in the crudest way to those existent in the alimentary tract. At the same time, if complete peptonization is characteristic of pepsin-proteolysis in the stomach, and failure to obtain such results in an artificial digestion is due to lack of withdrawal of the diffusible products formed, then certainly the experiments carried on in dialyzer tubes, with abundant opportunity for diffusion, and with a large excess of free hydrochloric acid, should show some indications of increased peptone-formation. But none were obtained.
It is more than probable that the rate of absorption of diffusible products from the stomach has been overestimated. Lea,144for example, assumes that, “normally the products of digestion, whether proteid or carbohydrate, are never met with in either the stomach or intestine in other than the smallest amounts, frequently to be described as merely traces.” This certainly implies a far more rapid absorption of proteoses and peptones from thestomach than results seem to justify. Indeed, recent facts obtained by Brandl,145working under Tappeiner’s direction, tend to show that absorption from the stomach is, under some circumstances at least, comparatively slow. Brandl’s experiments were conducted on large and vigorous dogs with gastric fistulæ, the stomach being shut off from the intestine by the simple introduction of a small rubber balloon into the pylorus, which when dilated completely closed the orifice. By carefully conducted experiments, it was shown that pure peptone, entirely free from proteose, is absorbed from the empty stomach in proportion to the concentration of the peptone solution. Thus, 7.5 grammes of peptone dissolved in water in such proportion as to make a five per cent. solution, and allowed to remain in the stomach for two hours, lost by absorption only 0.28 gramme, equal to 2.68 per cent. of the peptone introduced. Under similar conditions, a ten per cent. aqueous solution of peptone lost only 4.5 per cent. by absorption. On the other hand, when peptone was introduced in larger quantity, viz., in a twenty per cent. solution, absorption amounted to thirteen per cent. in two hours.
It is thus evident that pure peptones, even when taken into the stomach in fairly large amounts, and under conditions very favorable for rapid absorption, pass into the circulating blood very slowly. Obviously, however, one must not lose sight of the fact that when digestion is under way and the volume of blood consequently increased, there may be a corresponding rise in the rate of absorption. There is perhaps a hint of this conclusion in the influence of alcohol on the absorption of peptone as brought out by some of Brandl’s experiments. Thus, it was found that when alcohol was added in considerable quantity to a tenper cent. solution of peptone, the stomach-mucosa was greatly reddened, while in two hours the absorption of peptone amounted to 11.8 per cent. But in any event, these results certainly do not favor the view that the products of gastric digestion are absorbed as soon as they are formed. It is, no doubt, quite different in the intestine, but in the stomach, where pepsin-proteolysis occurs, we have, I think, no grounds for assuming that either peptones or proteoses are rapidly absorbed. Hence, it might perhaps be considered that the results of pepsin-proteolysis in the living stomach are much the same as those obtained in artificial digestion experiments.
Still, there are other differences between natural digestion and artificial proteolysis than those connected with the possible absorption of the more diffusible products of digestion. Thus, in the living stomach there is an ever-increasing secretion of hydrochloric acid, and perhaps also of pepsin, more or less proportional to the extent of proteolysis. On this point Brandl’s experiments again give us some light. Thus, it was found that the introduction of an aqueous solution of peptone into the empty stomach led to the secretion of an acid fluid containing on an average 0.24 per cent. HCl, while, under similar conditions, the introduction of sugar or potassium iodide was followed by the secretion of a fluid containing on an average only 0.13 per cent. HCl. Further, the absolute amount of acid found after the introduction of peptone was far greater than when sugar or iodide was introduced, since peptone led to an increase of at least fifty per cent. in the volume of fluid secreted. Hence, proteolysis in the living stomach may give rise to such an increased production and secretion of hydrochloric acid that formation of the terminal products of gastric digestion may be greatly accelerated. That such in fact is the case, I have no manner of doubt,but that it may result in the complete conversion of the so-called primary and secondary proteoses into peptone I very much question. In fact, such examinations as I have made of the stomach-contents after a suitable test-meal have always resulted in the finding of a relatively large amount of proteoses. To be sure, true peptone may be detected and in fairly large amounts, but whenever a quantitative determination of the relative proportion of the two has been made, the proteoses have always been in excess. I have already reported elsewhere the results of some experiments in this direction made on a healthy young man, where the stomach-contents were withdrawn at varying periods after the ingestion of weighed amounts of coagulated egg-albumin. Thus, in oneexperiment146the stomach was thoroughly rinsed with water, after which 138 grammes of finely divided coagulated-albumin, equal to 16 grammes of dry albumin, were ingested. Three-quarters of an hour thereafter, the stomach-contents were withdrawn by lavage and analyzed. As a result, 1.41 grammes of albumoses were separated and weighed, and 0.84 gramme of peptones, the relative proportion being expressed by sixty-two per cent. of albumoses and thirty-seven per cent. of peptones, calculated on the 2.25 grammes of soluble products recovered. This expresses the general character of the results obtained in experiments of this nature, and in my opinion adds emphasis to the statement already made, that complete peptonization is not a feature of pepsin-proteolysis, either in the artificial or in the natural process as it takes place in the living stomach.
Gastric digestion is to be considered rather as a preliminary step in proteolysis, preparatory to the more profound changes characteristic of pancreatic digestion, in which the ferment trypsin is the important factor. We can thus seehow, as in the case of Czerny’s dogs, an animal may be perfectly nourished without a stomach, digestive proteolysis being carried on solely by the pancreatic fluid. You will remember that two of the dogs operated on by Czerny and his pupils lived between four and five years after the operation, with the stomach completely removed, and yet during this period they were well nourished and ate all varieties of food with apparently a normal appetite.147Evidently, then, in some cases at least, digestive proteolysis can be carried on without this preliminary action of the gastric juice.Ogata148arrived at essentially the same conclusion by the establishment of a duodenal fistula, shutting off the stomach from the intestine by means of a small rubber ball which could be inflated with water. On then introducing coagulated egg-albumin and other forms of proteid matter into the duodenum, he found that digestion was at least sufficiently complete to satisfy all the demands of the system. The only unsatisfactory result was with collagenous foods, which plainly showed the need of a preliminary acid digestion. More recently still, Cawallo and Pachon,149working in Richet’s laboratory, have studied the digestibility of different kinds of proteid foods in a dog, upon which they had performed a gastrectomy; the entire fundus, as well as the pyloric portion, of the stomach having been removed. In an animal so operated upon, after recovery was complete, solid food, as meat, was completely digested when taken in small quantities at a time. Raw meat, however, was less completely utilized, the fæces showing portions of undigested fibres. Still, it was apparent that intestinal digestion alone was capable ofaccomplishing all that was necessary for the complete nourishment of the animal, when it had once become accustomed to the changed condition of its alimentary tract.
These facts are cited not to belittle the importance of gastric digestion in the nutrition of the body, but rather to emphasize the probability that pepsin-proteolysis is simply a preliminary step in digestion; that its function is not in the direction of a complete peptonization of the proteid foods ingested, but that its action is especially directed to the production of soluble products, proteoses, which can be further digested in the small intestine, or perhaps directly absorbed after they have passed through the pylorus, or even from the stomach itself to a certain extent.
It is very evident from what has been said that all forms of proteid matter,i.e., all the members of the three main groups spoken of in our classification of the proteids, excepting only nuclein, reticulin, and the keratins, are capable of undergoing proteolysis with pepsin-hydrochloric acid. Further, in every case the main products of the transformation are proteoses; viz., albumoses, caseoses, gelatoses, vitelloses, myosinoses, etc., according to the nature of the proteid undergoing proteolysis; true peptones being formed in less abundance. Corresponding to each of these groups are primary and secondary proteoses, all possessed of many points in common, both chemical and physiological, yet differing from each other in many minor respects. These are the important products of gastric digestion, of pepsin-proteolysis, and it may be wellto consider for a moment some of the physiological properties of the proteoses and of peptones as well, in order that we may the better comprehend the general nature of these substances with reference to their possible action in the economy.
As far back as 1880, Schmidt-Mülheim150discovered that the injection of aqueous solutions of peptone into the blood-vessels of living dogs was attended by a series of remarkable phenomena. Thus, the animal passed at once into a condition of narcosis resembling that produced by chloroform, accompanied by a fall of general blood-pressure so great that the animal was liable to die, as from asphyxia. Further, there was evidence of some marked change in the condition of the blood, as indicated by loss of the power of spontaneous coagulation, while the peptone itself evidently underwent some alteration, or else was rapidly eliminated, since it could not be detected in the blood a short time after its introduction. These experiments, however, were not conducted with true peptone but with Witte’s “peptonum siccum,” which at that time, at least, was composed in great part of proteoses. The general character of these interesting results was confirmed by Fano,151who found that the injection of so-called peptone in the proportion of 0.3 gramme per kilo. of body-weight was sufficient to bring about complete narcosis, together with loss of coagulability on the part of the blood. Very suggestive, however, was the fact that Fano, on trying similar experiments with the peptone formed by pancreatic digestion, viz., with antipeptone, which presumably contained a far smaller proportion of proteoses, failed to obtain like results; the tryptone, so-called, being exceedingly irregular in its action, in many cases producing no effect whatever.
The discovery at this date of the several albumoses, and their presence in large amounts in all so-called peptones, led to a study of their physiological action with special reference to the observations of Schmidt-Mülheim and Fano. Politzer,152working under Kühne’s guidance, was the first to experiment in this direction, and his results are full of interest as throwing light on the action of the individual albumoses. Thus proto, hetero, and deuteroalbumose are all active physiologically, giving rise when injected into the veins of dogs and cats to strong narcotic action, varying somewhat in intensity in different individuals. There is also produced a marked fall in blood-pressure, due apparently to vaso-motor paralysis, the action being manifested chiefly, if not wholly, on the splanchnic region. Thus, after an injection of one of these albumoses, the mesenteric vessels are always strongly congested, accompanied frequently by the appearance of a bloody serum in the peritoneal cavity. Narcotic action is manifested only so long as the blood-pressure remains sub-normal, and is due presumably to this marked accumulation of blood in the large abdominal veins, thus leading to anæmia of the brain. Albumoses and peptones injected into the jugular vein likewise produce fever, presumably through some action on the nervous system by which the equilibrium of tissue-metamorphosis is interfered with.153
Further, Politzer found that all of the albumoses either delayed or prevented altogether the coagulation of the blood, in conformity with the observations of Schmidt-Mülheim and Fano. In all of these actions the primaryalbumoses appeared most effective, deuteroalbumose least so. Heteroalbumose, however, was constantly most active, especially in delaying the coagulation of the blood. With amphopeptone, there was far less narcosis and less diminution of blood-pressure, while the effect on the coagulability of the blood was more or less variable, frequently being entirely negative. Antipeptone, on the other hand, was found almost wholly wanting in any constant effects, although in one instance deep narcosis was produced. Thus, from Politzer’s experiments, it was made clear that the albumoses, when introduced directly into the blood-current, possess a far greater toxic action than either amphopeptone or antipeptone. Albumoses, in sufficiently large doses, were invariably fatal, while peptones never produced fatal results so long as the kidneys of the animal remained intact. The extreme solubility and diffusibility of peptones, coupled perhaps with their marked diuretic action, lead to rapid elimination through the kidneys, and their consequent removal from the system.
Many of these observations made with the albumoses I have repeated with several of the proteoses and peptones more recently studied, as protocaseose, protoelastose, the globuloses, and others. The results may be taken as practically confirmatory of the older observations, and I make mention of them in this general way simply to emphasize the fact that all of the proteoses, though perhaps showing individual peculiarities, are possessed of marked physiological properties, which plainly testify to their toxic nature, when introduced directly into the blood-current.
Young animals are particularly sensitive to the injection of proteoses into the blood, even when the introduction takes place very gradually.154Thus, a young, healthy dogof 2 kilos. body-weight, eight weeks old, died in one hour after the injection into the jugular vein of 1 gramme of protoalbumose in 20 c.c. of water, thus affording a good illustration of the extreme toxicity of this albumose when introduced directly into the blood.
Of greater interest, physiologically, are the changes the individual proteoses undergo after their injection into the blood. As already stated, peptone so injected may appear in the urine wholly unaltered. Thus,Neumeister155has made injections of both amphopeptone and antipeptone in the case of dogs, and was able to detect the peptone very quickly in the urine. I have made like experiments with other forms of peptone and obtained similar results; thus, a pure amphopeptone formed from casein by pepsin-proteolysis (2 grammes in 15 c.c. water) was injected into the jugular vein of a dog weighing 5 kilos. The urine collected during several hours after the injection was heated to boiling, and saturated while hot with ammonium sulphate. The filtrate, on being tested with cupric sulphate and potassium hydroxide, gave a fairly strong biuret reaction for peptone. Another similar experiment made with antipeptone, formed from the myosin of muscle-tissue, gave like results.
With proteoses, however, different results are obtained, asNeumeister156first pointed out. These bodies introduced into the blood undergo more or less of a change prior to their excretion in the urine, the change partaking of the character of a hydrolytic cleavage in which the primary proteoses are transformed into secondary proteoses, while deuteroproteoses are changed into peptones. This is not necessarily to be interpreted as meaning that the full equivalent of the proteose injected appears in the urine, but that the portion which is eliminated through the kidneys tends to undergo a transformation somewhereen route, akin to the change produced in pepsin-proteolysis. As to how common or complete this transformation is under the above circumstances, we have no positive knowledge. Such a hydrolytic change certainly occurs in the case of the dog, and the experimental evidence is in favor of the view that the transformation is effected in the kidneys by the pepsin secreted through the urinary tubules, where there is momentarily a formation of free acid. In the rabbit, on the other hand, no such change occurs; the urine from this animal contains practically no pepsin, and consequently the proteoses eliminated through the kidneys are excreted unaltered. As, however, the experiments ofStadelmann157and others have shown that the urine of all carnivora, and of man as well, contains a ferment which, on the addition of a suitable amount of hydrochloric acid, will digest fibrin with formation of the ordinary products of pepsin-proteolysis, it is to be presumed that all proteoses passing through the kidneys will undergo at least some change prior to their excretion in the urine.
However this may be, it is very evident that the proteoses formed in gastric digestion cannot be absorbed as such directly into the blood-current. Introduced into the blood, they behave in such a manner as to warrant the conclusion that they are truly foreign substances, and the system makes a brave endeavor to remove them as speedily as possible. The same may be said of amphopeptones, from which we may conclude that all of these products of pepsin-proteolysis undergo some transformation during the process of absorption, by which their toxicity is destroyed and their nutritive qualities rendered fully available for the needs of the body. Discussion of this question, however, will be left until the next lecture.
In view of these pronounced physiological properties of the proteoses, it is interesting to recall the now well-known fact that many of the chemical poisons produced by bacteria are proteose-like bodies, chemically, at least, closely akin to the proteoses resulting from pepsin-proteolysis. Thus,Wooldridge158as early as 1888 pointed out that an alkaline solution of tissue-fibrinogen exposed to the action of anthrax-bacilli suffered some change, so that when introduced into the blood it possessed the power of producing immunity to anthrax. This observation was verified by Hankin,159who further showed that the substance formed by the anthrax-bacilli was a veritable albumose, and that it truly possessed the power of producing immunity. SidneyMartin160carried the matter still further, and by growing the anthrax-bacilli in a pure solution of alkali-albuminate prepared from blood-serum, proved the formation of both primary and secondary albumoses, as well as of peptone, leucin, tyrosin, and a peculiar alkaloidal substance of pronounced toxic properties. Martin finds that the albumoses are not as poisonous as the alkaloid, and surmises that the alkaloid is contained in the albumose molecule in the nascent state; further, he suggests that the albumoses in small doses may exert some protective influence, while in larger doses they act as vigorous poisons. How true this may be I cannot say, but my own experience convinces me that the anthrax-bacilli grown in a culture medium composed of alkali-albuminate, prepared from egg-albumin, to which the necessary inorganic salts and some glycerin have been added, do give rise to albumoses and peptones which are truly endowed with toxic properties.
Albumose-like bodies have also been obtained by BriegerandFränkel161with the bacillus of diphtheria. These, too, were endowed with powerful poisonous properties, and when introduced into the tissues of the body gave rise to reactions resembling those produced by the Löffler bacillus. In my own laboratory, recent experiments made with the bacillus of glanders have shown that when grown in a slightly acid medium containing alkali-albuminate, albumoses, peptones, and crystalline bodies such as leucin and tyrosin are formed in considerable quantities.Kresling162has reported similar results. With the tubercle-bacilli, many like results have been recorded. Thus, among others, Crookshank andHerroun163have reported the finding of albumoses, peptone, and a ptomaine when the bacilli have been grown in glycerin agar-agar, and also in fluid media.
Koch164has made a special study of the albumose which he considers as the specific toxic agent of the so-called tuberculin. This albumose was found by Brieger andProskauer165to have a somewhat peculiar composition, inasmuch as it contains forty-seven to forty-eight per cent. of carbon and only 14.73 per cent. of nitrogen, agreeing, however, in this respect very closely with the peptone formed from egg-albumin by the action of bromelin.166Still more recently,Kühne167has made a thorough study of this albumose, as well as of the other products elaborated by the growth of the tubercle-bacillus. He designates all of the peculiar albumoses formed by these bacilli asacrooalbumoses. They are endowed with marked chemical andphysiological properties, causing a rise of temperature when injected into the blood, as well as other phenomena more or less pronounced. It is thus evident there is ample ground for the statement that all nutritive media in which pathogenic bacteria have been planted are liable to contain, sooner or later, toxic substances, many of which at least are closely related to, if not identical with, the albumoses. It is not my purpose, however, to consider these points in detail, nor to quote the many results obtained by other workers in this direction.
I wish merely to call attention to the fact that the proteoses, and likewise the peptones formed by pepsin-proteolysis, are more or less toxic when introduced directly into the blood, and that they share this property with the proteoses formed by bacterial organisms, or by the enzymes which they give rise to. In other words, these primary cleavage or alteration products of the proteid molecule, however produced, are more or less poisonous, and if introduced into the blood-current without undergoing previous change may show marked physiological action. It is, of course, not to be understood that these bodies are all alike. They are surely closely related and possess many points in common, especially so far as their chemical properties are concerned, but their chemical constitution and their physiological action must vary more or less with their mode of origin.
In any event, it is very evident that the proteoses and peptones formed in the alimentary tract by pepsin-proteolysis must undergo some transformation, before reaching the blood-current, by which their peculiar physiological properties are modified. This modification may be associated with a conversion into the serum-albumin, or globulin of the blood. However this may be, the fact remains that these proteoses formed so abundantly during digestion can be absorbed and serve as nutriment for the animalbody, but between their formation as a result of proteolysis and their passage into the blood they are exposed to some agency, or agencies, doubtless in the very act of absorption, by which a further transformation is accomplished. With this point we shall be able to deal more in detail in the next lecture.