LECTURE II. PROTEOLYSIS BY PEPSIN-HYDROCHLORIC ACID, WITH A CONSIDERATION OF THE GENERAL NATURE OF PROTEOSES AND PEPTONES. PROTEOLYSIS BY PEPSIN-ACID.

Gastric digestion is essentially an acid digestion. As a proteolytic agent, pepsin can act only in the presence of acid, and we have every reason for believing that the enzyme and the acid form a compound, which in turn combines with the proteid undergoing digestion; or, what amounts to much the same thing, that the acid perhaps forms first a compound with the proteid, to which the pepsin can then unite to form a still more complex compound capable of undergoing hydration and cleavage. Pepsin-proteolysis, therefore, is strictly the proteolysis produced by pepsin-acid. In view of this fact, we may well give a moment’s thought to the nature and origin of this acid.

Without attempting any statement of the gradual development of our knowledge regarding the acid of the gastric juice, we may accept the now well-established fact that the acid is hydrochloric acid, and that it has its origin in the parietal, or so-called border-cells of the gastric glands. That the acid is derived from the decomposition of chlorides is practically self-evident, but Cahn94 has added experimental proof which removes all shadow of doubt, through his study of the gastric secretion in animals deprived for many days of salt; the gastric juice in such cases being perfectly neutral in reaction, but normal as regards its content of pepsin.

The way in which the specific gland-cells manufacture free hydrochloric acid out of material contained in an alkaline medium is somewhat doubtful. There are, however, at the present day two theories worthy of special notice. The first is based upon observations made by Maly95 many years ago, which tend to show that certain mineral salts present in the blood are capable of reacting upon each other with formation of hydrochloric acid. Thus, while the blood is an alkaline fluid, it really owes its alkalinity to the presence of two acid salts, viz., sodium bicarbonate (HNaCO₃) and disodium hydrogen phosphate (HNa₂PO₄). This latter compound, acted upon by the carbonic acid of the blood, is transformed into a dihydrogen sodium phosphate with simultaneous formation of acid sodium carbonate, as shown in the following equation:

Na₂HPO₄ + CO₂ + H₂O = NaH₂PO₄ + HNaCO₃.

This acid sodium phosphate dissolved in a fluid containing sodium chloride, gives rise to free hydrochloric acid by a very simple reaction:

NaH₂PO₄ + NaCl = Na₂HPO₄ + HCl.

It is also to be noted that the disodium hydrogen phosphate, may, likewise, give rise to hydrochloric acid through its action on calcium chloride, as indicated by the following equation:

2Na₂HPO₄ + 3CaCl₂ = Ca₃(PO₄)₂ + 4NaCl + 2HCl.

It is thus evident that hydrochloric acid may originate in the inter-reaction of these several salts which are known to be present in the blood; but obviously, the above reactions cannot take place in the blood itself, and we must look to the selective power of the epithelial cells of the gastric glands, as suggested by Gamgee,96 for the withdrawal of the needed salts from the blood. Once present in the acid-forming cells, and perhaps aided by the inherent qualities of the protoplasm, the necessary chemical reactions may be assumed to take place, after which the newly formed acid may pass from the gland-cells into the secretion of the gland.

A later theory regarding the formation of the acid of the gastric juice emanates from Liebermann.97 This investigator claims the existence in the mucous membrane of the stomach of an acid-reacting, nuclein-like body, which is apparently a combination of the phosphorized substance lecithin with a proteid. To this compound body Liebermann gives the name of lecithalbumin. It is apparently located in the nuclei of the gastric cells, is strongly acid in reaction, and, according to Liebermann, is an important agent in the production of the free hydrochloric acid of the gastric juice, although its action is somewhat indirect. According to this theory, the free acid is formed in the mucous membrane of the stomach from sodium chloride, through the dissociating action of the carbonic acid coming from normal oxidation. The thus-formed acid then diffuses in both directions, viz., through the lumen of the gland into the stomach-cavity, and in part in the opposite direction into the veins and lymphatics. It is the assumed function of the lecithalbumin to react with the alkaline sodium carbonate, produced simultaneously with the hydrochloric acid. This naturally gives rise to the liberation of carbonic acid and to the formation of a non-diffusible sodium-lecithalbumin compound, which is retained for the time being in the body of the cell. When the circulation of the blood, accelerated by the digestive process, returns to its ordinary pace, this latter compound is slowly decomposed by the carbonic acid with formation of the readily diffusible sodium carbonate, which passes into the blood-current. The rate of this latter reaction is impeded, or, perhaps regulated, by the swelling up of the lecithalbumin-containing cells, thus rendering the imbibition of the carbonic acid a slow process. The rate of production of the hydrochloric acid by this hypothetical process depends primarily upon the blood supply, and the oxidative changes by which carbonic acid is formed.

There is much that might be said for and against this theory,98 but we cannot stop to discuss it here. Like the previous theory, it implies the production of hydrochloric acid from a chloride or chlorides, through chemical processes taking place in the stomach-mucosa, and presumably in the large border-cells of the peptic glands. This hydrochloric acid, as you know, in the act of secretion, reacts upon the pepsinogen with which it may come in contact, transforming it into pepsin. It also has the power of combining with all forms of proteid matter, not excepting the products of proteolytic action, to form acid compounds in which the so-combined acid, although equal quantitatively to the original amount of free acid, is less active in many ways. Thus, it does not possess in the same degree a destructive action on the amylolytic ferments;99 it does not play the same part in aiding the proteolytic action of pepsin, and its antiseptic power is far from equal to that of a like amount of free acid.100

With relatively large amounts of proteid, we may have half or even quarter saturated proteid molecules, in which the weakness of the combined acid is far more pronounced than in the case of the fully saturated molecule. Such a condition of things must obviously exist in the early stages of gastric digestion. With an excess of proteid matter in the stomach, some time must elapse before the secretion of hydrochloric acid will be sufficient to furnish acid for all of the proteid matter present, yet pepsin-proteolysis does not wait the appearance of free acid. Indeed, the proteid matter may not have combined with more than half its complement of hydrochloric acid before digestive proteolysis is well under way. I have made many analyses of the stomach-contents after test meals, and under other conditions, where no free acid could be detected by the tropaeolin test, or better, by GÜnzburg’s reagent (phloroglucin-vanillin), although phenol­phthalein as well as litmus showed strong acid reaction, and yet not only could acid-albumin be detected in the filtered fluid, but likewise proteoses and peptones. In other words, pepsin-proteolysis can proceed in the absence of free hydrochloric acid, although not at the same pace. Hence, proteoses and even peptones may make their appearance in the stomach-contents at a very early period of digestion, i.e., the final products of proteolysis may be found in a mixture containing even a large proportion of wholly unaltered proteid, and obviously at an early stage in the process. Expressed in other language, a portion of the first formed acid-albumin or syntonin may be carried forward by the digestive process to the secondary proteose and peptone stage, before the larger portion of the ingested proteid food has even combined with sufficient acid to insure the complete formation of acid-albumin. This introduces another factor, to be referred to later on, viz., the relative combining power of different forms of proteid matter, especially the proteoses and peptones, as contrasted with native proteids.

In proof of the statement that pepsin-proteolysis can proceed in the absence of free hydrochloric acid, provided combined acid be present, allow me to cite one or two experiments bearing on this point. A perfectly neutral solution of egg-albumen, containing 0.8169 gramme of ash-free albumin per 10c.c. of fluid, was employed as the proteid material. In order to completely saturate the proteid contained in 20c.c. of this neutral albumen solution, 50c.c. of 0.2 per cent. HCl were required. Two mixtures were then prepared as follows:

A. Twenty c.c. of the neutral albumen solution + 50c.c. 0.2 per cent. HCl + 30c.c. of a weak aqueous solution of pepsin, perfectly neutral to litmus. This mixture gave only the faintest tinge of a reaction for free acid when tested by GÜnzburg’s reagent.

B. Twenty c.c. of the neutral albumen solution + 25c.c. 0.2 per cent. HCl + 30c.c. of the neutral pepsin solution. In this mixture, the proteid matter was obviously only half saturated with acid.

The two solutions were placed in a bath at 40°C., where they were allowed to remain for forty-four hours, a little thymol being added to guard against any possible putrefactive changes. At the end of this time the amount of undigested albumin was accurately determined. The 20c.c. of original albumen solution contained 1.6338 grammes of dry coagulable albumin. At the end of the forty-four hours, A contained only 0.5430 gramme of unaltered albumin, or acid-albumin, while B contained 1.2225 grammes. That is to say, in the mixture A, where the acid existed wholly in the form of combined acid, but with the albumin completely saturated, 1.0908 grammes of the proteid were converted into soluble albumoses and peptones. In B, on the other hand, where the albumin was only half saturated with acid, 0.4113 gramme of the proteid was converted into soluble products. This difference in action is made more striking by the statement that where the proteid was only half saturated with acid, 25.1 per cent. of the albumin was digested; while with a complete saturation of the proteid, 66.7 per cent. of the albumin was digested.

To give emphasis to this matter, a second experiment may be quoted as follows: The proteid used was the same neutral solution of egg-albumen containing 0.8169 gramme of albumin per 10c.c. Two mixtures were prepared as follows:

A. Ten c.c. of the neutral albumen solution + 21.7c.c. 0.2 per cent. HCl, the amount needed to completely saturate the proteid, + 40c.c. of a weak solution of pepsin, perfectly neutral.

B. Ten c.c. of the albumen solution + 10.9c.c. 0.2 per cent. HCl + 40c.c. of the pepsin solution, making a mixture half saturated with acid.

These two solutions were warmed at 40°C. for seventeen hours. The extent of digestive action was then determined, when it was found that in A only 0.1638 gramme of the proteid was undigested, while in B, 0.6088 gramme remained unaltered. In other words, where the proteid was completely saturated with acid, but with an utter lack of free acid, 79.9 per cent. of the albumin was converted into albumoses and peptone, while in the mixture half saturated with acid only 25.4 per cent. was digested.

These two experiments thus give striking proof that free acid is not absolutely essential for pepsin-proteolysis. Digestion is, to be sure, more rapid and complete when free hydrochloric acid is present, but proteolysis is still possible, and even vigorous, when there is a marked deficiency of free acid. Further, as we have seen, proteolysis may proceed to a certain extent even though the amount of acid available is not sufficient to combine with more than half the proteid matter present.

These facts at once raise the question whether the products of proteolysis may not have a stronger affinity for acid than the native proteids; an affinity so strong that they may be able to withdraw acid from the acid-albumin first formed. One of our conceptions regarding pepsin-proteolysis is that acid is necessary for every step in the proteolytic process. A primary albumose, for example, cannot be further changed by pepsin, unless there is acid present for it to combine with. This being true, it is clear, in view of the fact that even peptones may appear in a digestive mixture containing an amount of acid insufficient to combine even with the albumin present, that the products of proteolysis must withdraw acid from the acid-albumin first formed. In regard to the first point, my own experiments certainly tend to show that the products of gastric digestion do combine with larger amounts of hydrochloric acid than undigested proteids; and further, that of the several products of proteolysis, the secondary proteoses combine with a larger percentage of acid than the primary proteoses, while true peptones combine with still larger amounts. In other words, the simpler and more soluble the proteid, the larger the amount of acid it is capable of combining with; a statement which accords with results obtained by other workers101 in this direction. Further, another factor of considerable importance in connection with the natural digestive process is that a dissolved proteid, such as protoalbumose for example, will combine more readily with free acid than an insoluble proteid; from which Gillespie102 is led to infer that in pepsin-proteolysis where there is no free acid present, only acid-albumin, proteoses may be formed to a limited extent at the expense of some of the acid of the acid-albumin, a portion of the latter being perhaps reconverted into albumin. The ability of the proteoses, however, to withdraw acid from its combination with a native proteid is perhaps best indicated by Kossler’s103 experiments, which show that a solution of acid-albumin containing only enough hydrochloric acid to hold the albumin dissolved, on being warmed at 40°C. for some hours with addition of a neutral solution of pepsin, may undergo partial conversion into albumose or peptone.

In spite of these facts, there is some evidence that while proteoses and peptones have the power of combining with more acid than a like weight of native proteid, the latter, leaving out all action of the pepsin, has a stronger affinity for the acid; in fact, the firmness or strength of the union appears to diminish as the products become simpler.104 Hence, a peptone separated from a digestive mixture, will part with its combined acid somewhat more readily than acid-albumin for example, although on this point there is not complete unanimity of opinion.105 In digestive proteolysis, however, where the pepsin is accompanied by a minimal amount of hydrochloric acid, insufficient perhaps to even half saturate the proteid present, the formation of proteoses and peptones must be accompanied by a withdrawal of acid from its combination with the native proteid.

In illustration of some of these points, and especially of the statement that the products of gastric digestion have the power of combining with more hydrochloric acid than the original proteid, allow me to cite the following experiment: 10c.c. of a neutral solution of egg-albumen containing about 0.82 gramme of pure dry albumin, free from mineral salts, required 23.8c.c. of 0.2 per cent. hydrochloric acid to completely saturate the proteid matter. A mixture was then prepared as follows: 10c.c. of the albumen + 24c.c. 0.2 per cent. HCl + 30c.c. of a neutral pepsin solution, the mixture showing a faint trace of free acid when tested by GÜnzburg’s reagent. This solution was placed in a thermostat at 38°C., and from time to time a drop of the fluid was removed and tested for free acid. If no reaction could be obtained, 0.2 per cent. hydrochloric acid was added to the mixture, until GÜnzburg’s reagent showed free acid to be again present. The following table shows the rate of disappearance of free acid, and the amounts of 0.2 per cent. HCl required to make good the deficiency. The mixture was placed at 38°C. on February 6th, at 11.30 A.M., and, as stated, contained a trace of free acid, 24c.c. 0.2 per cent. HCl having been added to accomplish this result.

From these results several interesting conclusions may be drawn, in conformity with the statements already made. Thus, as soon as proteolysis commences, the products formed begin to show their greater affinity for acid by withdrawing acid from its combination with the native proteid, a supposition which is necessary to account for even the starting of the proteolytic process. Further, it is evident that proteoses and peptones combine with a far larger equivalent of acid than the native albumin is capable of; 17c.c. of 0.2 per cent. HCl being required in the above experiment to satisfy the greater combining power of the newly formed products. This doubtless depends upon the cleavage of the large proteid molecule into a number of smaller or simpler molecules, each of the latter, perhaps, combining with a like number of HCl molecules. This view of the relationship of the individual proteoses and peptones is one more or less generally held, and is supported by many facts.106 However this may be, it is evident that the products of pepsin-proteolysis combine with a larger amount of hydrochloric acid than the mother-proteid, and that the transformation of the latter, at least under the conditions of this experiment, is a slow and gradual process. In the living stomach, on the other hand, where the secretion of acid is progressing with ever-increasing rapidity, it is easy to see that the process of proteolysis would naturally be much more rapid.

Just here we may recall the theory advanced by Richet107 quite a number of years ago that the acid of the gastric juice is a conjugate acid, composed of leucin and hydrochloric acid, a theory which has found little acceptance. Klemperer,108 however, assumed that solutions of leucin hydrochloride with pepsin would not digest albumin, but Salkowski and Kumagawa109 have shown by experiments that leucin and other amido-acids, as glycocoll, may be dissolved in hydrochloric acid in such proportion that the solution is practically composed of leucin hydrochloride, without interfering with the digestive action of pepsin-acid on blood-fibrin; the solution being physio­logically active, although GÜnzburg’s reagent shows an entirely negative result for free acid. If the matter is studied quantitatively, however, it will be found that the amido-acids combining in this manner with the hydrochloric acid of the gastric juice do give rise to some disturbance of proteolytic action;110 i.e., digestion may be less rapid, especially on egg-albumin, a conclusion which Salkowski111 has lately confirmed. Still, under such circumstances, digestion does go on and at a fairly rapid rate; hence, if there is a combination between the acid and these organic bodies, as is indicated by GÜnzburg’s reagent, the acid is still active physio­logically, even more so than in the compound formed by the interaction of proteid and acid. In other words, many of these neutral organic bodies that may originate in the stomach through fermentative processes, or otherwise, and which tend to combine with the acid of the gastric juice, do not, as a rule, impede pepsin-proteolysis to the same extent that an excess of proteid matter may. In fact, in artificial digestions long continued, pepsin-acid solutions containing considerable leucin, for example, may accomplish as much in the way of digesting proteid matter as the same amount of pepsin-acid without leucin; but the inhibitory action of the amido-acid is there, and may be shown during the first few hours of the experiment, when less proteoses and peptones are formed than in the control experiment without leucin.

It is foreign to our subject to discuss here methods for the detection of so-called free and combined hydrochloric acid in the stomach-contents, or the special significance of such findings in health and disease. I cannot refrain, however, in connection with what has been said above concerning the proteolytic action of pepsin in the presence of combined acid, from saying a word concerning the usual deductions drawn from the absence of free acid in the stomach-contents. As Langermann112 has recently expressed it, we have methods for discriminating between free and combined acid; we can, moreover, determine the amount of free acid, but is it not equally important to be able to say something definite concerning the amount of combined acid in the stomach-contents? Even in the absence of free hydrochloric acid there may be a sufficient amount of HCl secreted to answer all the purposes of digestion, and yet at no time may there be any free acid present to be detected by the various color-tests ordinarily made use of. I am aware that in ordinary examinations of the stomach-contents after a test meal the results are essentially comparative, and possibly all that are necessary for clinical purposes. What I wish to emphasize, however, is that in order to pass conclusively upon tsufficiency or insufficiency of the gastric secretion, it is wise to know not only the total acidity of the stomach contents and whether there is free acid or not, but to know more about the amount of combined acid present. Thus, there is a natural tendency to divide the fluids withdrawn from the stomach into three groups, viz., those which contain free acid in moderate amount, those which contain free acid in excess, and those in which free acid is entirely absent; but in the latter group, there may be very marked differences in the amount of acid combined with the proteid and other material present. It appears to me that one of the questions to be answered is whether there is sufficient combined HCl present to meet all the requirements for digestion. If there is, that gastric juice may be just as normal as the one containing free mineral acid, and yet, according to our present tendencies, we should be inclined to call the juice containing no free acid abnormal, although there may be sufficient combined acid present to meet all the requirements for digestion. Hence, in examination of the stomach-contents, it is well to consider the use of those methods which tend to throw light upon the amount of combined acid present, for in my opinion it is only by a determination of the total amount of combined acid that we can arrive at a true estimate of the extent of the HCl deficiency. Obviously, in simple clinical examinations of the stomach-contents after a test meal, where proteid matter is not present in large amount, free acid may reasonably be expected to appear after a definite period; but in any event, it is well to remember that free hydrochloric acid is not absolutely indispensable for fairly vigorous proteolytic action, and that in the presence of moderate amounts of proteid matter a large quantity of acid is required to even saturate the albuminous material.

Consider for a moment the amount of acid a given weight of proteid will combine with, before a reaction for free acid can be obtained. Thus, Blum113 has stated that 100 grammes of dry fibrin will require 9.1 litres of 0.1 per cent. hydrochloric acid to completely saturate it. Hence, with a daily consumption of 100 grammes of proteid, there would be needed for gastric digestion 4.5 litres of 0.2 per cent. hydrochloric acid daily, and even this would not suffice to give any free acid, assuming that none of the acid is used over again. The results I have already given for egg-albumin tend to show that 1 gramme of pure albumin, free from inorganic salts, when dissolved in a moderate amount of water will combine with about 30c.c. of 0.2 per cent. hydrochloric acid. Consequently, on this basis, 100 grammes of dry egg-albumin will combine with 3 litres of 0.2 per cent. HCl, and not until this amount of acid has been added to such a mixture will reaction for free acid be obtained with GÜnzburg’s reagent. Hence we can easily see, in view of these figures, that the production of hydrochloric acid by the gastric glands may at times be very extensive, without the stomach-contents necessarily containing free acid.

While I am by no means willing to agree with Bunge114 that the chief importance of the acid of the gastric juice is its action as an antiseptic, I am decidedly of the opinion that the lack of free hydrochloric acid in the stomach-contents is more liable to cause disturbance through the consequent unchecked development of bacteria than through lack of proteolytic action, assuming, of course, the presence of a reasonable amount of combined HCl. The hydrochloric acid of the gastric juice unquestionably plays a very important part in checking the growth and development of many pathogenic bacteria, as well as of less poisonous organisms, which are taken into the mouth with the food. On all, or at least on nearly all of these organisms, hydrochloric acid exerts a far greater destructive action when free than when combined with proteid matter. As Cohn115 has plainly shown, both hydrochloric acid and pepsin-hydrochloric acid quickly hinder acetic- and lactic-acid fermentation, but when the acid is combined with peptone, for example, it is no longer able to exercise the same inhibitory influence. It is also important to note that the lactic-acid ferment is not so sensitive to hydrochloric acid as the acetic-acid ferment. Consequently, when lactic-acid fermentation is once developed a comparatively large amount of HCl is required to arrest it. Hence, as we all know, a diminished secretion of hydrochloric acid renders possible acid fermentation of the stomach-contents, as well as putrefactive changes which would not occur in the presence of free HCl, and which are very incompletely checked when the acid is over-saturated with proteid matter.

Pepsin-proteolysis, however, may proceed, to some extent, at least, even though a small amount only of combined acid is present. The combined acid, however, must be hydrochloric acid, if proteolysis is to be at all marked. To be sure, pepsin will act in the presence of lactic acid, as well as in the presence of other organic acids, and inorganic acids, likewise, but such action at the best is considerably weaker than the action of pepsin-hydrochloric acid.116

The ferment pepsin can exert its maximum action only in the presence of free hydrochloric acid. There must be sufficient HCl to combine with all of the proteid matter present, and the products of proteolysis as fast as they are formed, if digestion is to be rapid and attended with the formation of a large proportion of the final products of proteolysis. It is under such conditions that our study of pepsin-proteolysis is usually conducted. Further, it is to be remembered that our knowledge of the products of such proteolytic action depends almost entirely upon data accumulated by artificial digestive experiments. In no other way can we be absolutely certain of the conditions under which the proteolysis is accomplished, for it is a significant fact, perhaps plainly evident from what has already been said in the preceding lecture, that the character of the products resulting from ordinary proteolysis is dependent in great part upon the attendant circumstances. Thus, with a relatively small amount of acid, and perhaps also of pepsin, the initial products of proteolysis are especially prominent, while with an abundance of both pepsin and free acid, coupled with long-continued action, the final products predominate. Between these two extremes there are many possible variations, as was, I think, made clear in the previous lecture. At the same time, it is to be noticed that these differences are mainly differences in the proportion of the several products, rather than in the nature of the resultant bodies.

In a general way, the products of pepsin-proteolysis may be divided into three main groups, viz., bodies precipitated by neutralization and represented mainly by the so-called syntonin or acid-albumin; bodies precipitated by saturation of the neutralized fluid with ammonium sulphate and represented by proteoses; bodies non-precipitable by saturation with ammonium sulphate and represented by amphopeptones. The relationship of the individual products may be clearly seen from the following scheme, arranged after the plan suggested by Neumeister.

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 each other 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, acetic acid117 is added drop by drop as long as a precipitate results, 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, as KÜhne118 has 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 the true 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 can be 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,120 Herth,121 Henninger122 and 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 the primary products 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 cleavage products123 formed by the action of pepsin-hydrochloric acid from 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.

Proteolysis of Paraglobulin.126

Proteolysis of Coagulated Egg-albumin.

Proteolysis of Casein from Milk.

Proteolysis of Myosin from Muscle.131

Proteolysis of Elastin.132

Proteolysis of Gelatin.133

Proteolysis of Phytovitellin134 (Crystallized) from Squash Seed.

Proteolysis of Phytovitellin135 (Crystallized) from Hemp Seed.

Proteolysis of Glutenin136 from Wheat.

Proteolysis of Zein.137

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, doubtless dependent 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.138 This 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 dried in 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 of individual 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-continued period 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 final products. Take as an illustration some results140 obtained 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 of this influence by experiments141 conducted 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 proteoses formed 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 200c.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 as KÜhne142 has 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 Waymouth Reid143 has 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 weight to 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,144 for 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 the stomach than results seem to justify. Indeed, recent facts obtained by Brandl,145 working 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 ten per 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 one experiment146 the 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 see how, 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.147 Evidently, then, in some cases at least, digestive proteolysis can be carried on without this preliminary action of the gastric juice. Ogata148 arrived 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,149 working 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 of accomplishing 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.

SOME PHYSIOLOGICAL PROPERTIES OF PROTEOSES AND PEPTONES.

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 well to 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-MÜlheim150 discovered 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,151 who 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,152 working 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 primary albumoses 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.154 Thus, a young, healthy dog of 2 kilos. body-weight, eight weeks old, died in one hour after the injection into the jugular vein of 1 gramme of protoalbumose in 20c.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, Neumeister155 has 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 15c.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, as Neumeister156 first 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 somewhere en 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 of Stadelmann157 and 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, Wooldridge158 as 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,159 who further showed that the substance formed by the anthrax-bacilli was a veritable albumose, and that it truly possessed the power of producing immunity. Sidney Martin160 carried 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 Brieger and FrÄnkel161 with 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. Kresling162 has reported similar results. With the tubercle-bacilli, many like results have been recorded. Thus, among others, Crookshank and Herroun163 have reported the finding of albumoses, peptone, and a ptomaine when the bacilli have been grown in glycerin agar-agar, and also in fluid media.

Koch164 has 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 and Proskauer165 to 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.166 Still more recently, KÜhne167 has 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 as acrooalbumoses. They are endowed with marked chemical and physiological 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 animal body, 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.

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