Part 6

[105] See Sansoni: Beitrag zur kenntniss des Verhaltens der Salzsäure zu den Eiweisskörpern in Bezug auf die Chemische Untersuchung des Magensaftes. Berliner klin. Wochenschrift, 1893, Nos. 42 and 43.

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: 10 c.c. of a neutral solution of egg-albumen containing about 0.82 gramme of pure dry albumin, free from mineral salts, required 23.8 c.c. of 0.2 per cent. hydrochloric acid to completely saturate the proteid matter. A mixture was then prepared as follows: 10 c.c. of the albumen + 24 c.c. 0.2 per cent. HCl + 30 c.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, 24 c.c. 0.2 per cent. HCl having been added to accomplish this result.

Time. Acid added to show trace of free acid. February 6, 11.30 A.M. " 2.15 P.M. 4.5 c.c., 0.2 per cent. HCl. " 5.00 P.M. 1.0 " " " " February 7, 8.45 A.M. 3.0 " " " " " 2.00 P.M. 1.0 " " " " " 5.00 P.M. 1.5 " " " " February 8, 8.30 A.M. 1.0 " " " " " 2.30 P.M. 0.0 " " " " February 9, 8.30 A.M. 3.0 " " " " February 10, 9.30 A.M. 2.0 " " " " ---- 17.0

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; 17 c.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.

[106] See Gillespie: On the Gastric Digestion of Proteids. Journal of Anatomy and Physiology, vol. 27, p. 209.

Just here we may recall the theory advanced by Richet[107] 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 Kumagawa[109] 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 Salkowski[111] 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.

[107] Le suc gastrique chez l’homme et les animaux, ses propriétés chimiques et biologiques. Paris, 1878.

[108] Zeitschr. f. klin. Medicin, Band 14, Heft 1 and 2.

[109] Ueber den Begriff der freien und gebundenen Salzsäure im Magensaft. Virchow’s Archiv, Band 122, p. 235.

[110] Rosenheim: Centralbl. f. klin. Medicin, 1891, No. 39. F. A. Hofman, ibid., No. 42.

[111] Ueber die Bindung der Salzsäure durch Amidosäuren. Virchow’s Archiv, Band 127, p. 501.

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 Langermann[112] 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.

[112] Virchow’s Archiv, Band 128, p. 408.

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, Blum[113] 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 30 c.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.

[113] Zeitschr. f. klin. Medicin, Band 21, p. 558.

While I am by no means willing to agree with Bunge[114] 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 Cohn[115] 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.

[114] Physiologische und Pathologische Chemie, p. 153.

[115] Ueber die Einwirkung des künstlichen Magensaftes auf Essigsäure-und Milchsäure gährung. Zeitschr. f. physiol. Chemie, Band 14, p. 75. See also Hirschfeld: Pflüger’s Archiv f. Physiol., Band 47, p. 510.

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]

[116] Chittenden and Allen: Influence of Various Inorganic and Alkaloidal Salts on the Proteolytic Action of Pepsin-Hydrochloric Acid. Studies in Physiol. Chem., Yale University, vol. 1, pp. 91, 94.

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.

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 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 acid[117] 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.

[117] Saturated with sodium chloride.

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 Kühne[118] 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.

[118] Erfahrungen über Albumosen und Peptone. Zeitschr. f. Biol., Band 29, p. 2.

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]

[119] Kühne and Chittenden: Peptones. Studies in Physiol. Chem., Yale University, vol. 2, p. 14.