Coagulatedegg-albumin.Atmidalbumoseprecipitatedby NaCl.Atmidalbumoseprecipitated byNaCl + acid.Deutero-atmidalbumose.Antialbumid.C52.3355.1355.0451.9953.79H6.986.936.896.607.08N15.8414.2814.1713.2514.55S1.811.66....0.98....O23.0422.00....27.18....
Here we see that two of these primary albumoses formed by the action of superheated water, like the previously described antialbumid, show a loss of nitrogen with a marked increase in the content of carbon. Evidently, they are related to the antialbumid formed by the action of dilute acid. They are, however, soluble in water, and in many ways differ from true antialbumid, but there is evidently an inner relationship. The so-called deuteroatmidalbumose shows a still more noticeable falling off in nitrogen and sulphur, while the content of carbon is more closely allied to that of the mother-proteid. The albumose precipitable by sodium chloride, although different from an albumid, evidently comes from the anti-group and is a cleavage product which in turn may undergo further hydration and splitting by continued treatment. The so-called deutero-body, on the other hand, may well be a representative of the hemi-group.90
It is not my purpose here to enter into details connected with the action of superheated water on proteids. Such a course would take us too far from our present subject, but I do wish to emphasize the fact that even the most resistant of proteids has an innate tendency to undergo hydration and cleavage, and that even simple heating with water alone, at a temperature slightly above 100° C., is sufficientto induce at least partial solution of the proteid. Further, this solvent action in the case of water and dilute acids, at least, is certainly associated with marked chemical changes. It is not mere solution, it is not simply the formation of one soluble body, but solution of the proteid is accompanied by the appearance of a row of new products, in which the terminal bodies are crystalline substances of simple composition. Further, this conclusion does not rest upon the results obtained from a single proteid, for I have at various times studied also the primary products formed in the cleavage of casein, elastin, zein, and other proteids by the action of hot dilute acid, and in all cases have obtained evidence of the formation of several proteose-like bodies, as well as of true peptones.
By the action of more powerful hydrolytic agents, such as boiling hydrochloric acid to which a little stannous chloride has been added to prevent oxidation, the proteid molecule may be completely broken down into simple decomposition products, of which leucin, tyrosin, aspartic acid, glutamic acid, glucoprotein, lysin, and lysatinin are typical examples.91In other words, by this and other methods of treatment, which we cannot take time to consider, we can easily break down the albumin-molecule completely into bodies which, as we shall see later on, are typical end-products of trypsin-proteolysis, and which are far removed from the original proteid. But, as we have seen, even the primary bodies formed in the less profound hydrolysis induced by superheated water, do not show the composition of the mother-proteid. Hydration and cleavage leave their marks upon the products, and thereby we know that solution of the proteid is the result of something more than a mere rearrangement of the atoms in the molecule.
Further, we are to remember that boiling dilute acid and superheated water tend to produce a cleavage along specific lines; viz., a cleavage into the anti- and hemi-groups of the molecule, and as representatives of these groups we may, in the hydration of every native proteid, look for two distinct rows of closely related substances.
In digestive proteolysis it will be our purpose to show that cleavage of much the same order occurs, not necessarily resulting, however, in the formation of identically the same products, but certainly accompanied with the production of bodies belonging to the hemi- and anti-groups, although they may be less sharply separated from each other than in the cleavage with dilute sulphuric acid.
The body originally described as hemialbumose, and identified as a product of every gastric digestion, is now known to be a mixture of closely related substances ordinarily spoken of as albumoses,92or generically as proteoses. These are primary products in the digestion of every form of proteid matter, intermediate between the mother-proteid and the peptone which results from the further action of the proteolytic enzymes. Associated with the hemialbumoses are corresponding antialbumoses, coming from the anti-half of the proteid molecule, and differing from their neighbors, the hemi-bodies, mainly in their behavior toward the ferment trypsin. Thus, we have the counterpart of the many bodies described by Meissner, although now arranged systematically and on the basis of structural and other differences not thought of in his day.
By the initial action of pepsin-acid, proteids are first transformed into acid-albumin or syntonin, then, by the further action of the ferment, this body is changed into the primary proteoses, proto and heteroproteose, of each of which there must be two varieties, a hemi and an anti.These may then undergo further transformation into what is known as a secondary proteose, viz., deuteroproteose, of which there must likewise be two varieties, corresponding to the hemi- and anti-groups respectively. By continued proteolytic action there results as the final product of gastric digestion peptones; approximately, an equal mixture of so-called hemipeptone and antipeptone, generally known as amphopeptone. Such a peptone exposed to the proteolytic action of trypsin should obviously break down in part into simple crystalline bodies, leaving a residue of true antipeptone. In truth, this is exactly what does happen when the peptone resulting from gastric digestion is warmed with an alkaline solution of trypsin. The so-called hemipeptone quickly responds to the action of the pancreatic ferment, and is converted into other products, while the so-called antipeptone resists its action completely, thus giving results in harmony with our general conception of the proteid molecule.
Albumin Molecule.
(Hemi-groups.Anti-groups.)ProtoalbumoseHeteroalbumoseAntialbumid(amphoalbumose)(amphoalbumose)DeuteroalbumoseDeuteroalbumoseDeuteroalbumose(amphoalbumose)(amphoalbumose)(antialbumose)AmphopeptoneAmphopeptoneAntipeptone
On the basis of these facts, and others not yet mentioned, we may accept provisionally, at least, the above schematic view, suggested in part by Neumeister,93of thegeneral line of proteolysis as it occurs in pepsin-digestion; a view which clearly expresses the significant relationship of the hemi- and anti-groups in the proteid molecule.
The dark and light lines in this scheme are intended to represent the relative share which the hemi- and anti-groups take in the formation of the individual bodies. Thus, we see that protoproteoses have their origin mainly in the hemi-groups of the molecule, although, as the fine line indicates, anti-groups are somewhat concerned in their construction. Heteroproteoses, on the other hand, come mainly from the anti-groups, but still some hemi-groups have a part in their structure. As previously stated, these two primary proteoses by further hydrolytic action may be transformed into secondary products; viz., into deuteroproteoses, but, as the above scheme indicates, the two deutero bodies will be more or less unlike in their inner nature. In one sense, they are both amphodeuteroproteoses, but they necessarily differ in the proportion of hemi- and anti-groups they contain. By the still further action of pepsin-acid, the deutero bodies may be changed, in part at least, into peptone,i. e., into amphopeptone, although, as Neumeister has pointed out, protoproteose tends to yield an amphopeptone in which the hemi-groups predominate, while the peptone coming from heteroproteose contains an excess of anti-groups. Moreover, in the gastric digestion of any simple proteid a certain number of anti-groups are split off in the form of antialbumid, a body which is only slowly digestible in pepsin-acid. By the very powerful proteolytic action of a strong gastric juice, however, antialbumid may be somewhat digested, and is then transformed into antideuteroalbumose, which in turn may be eventually changed into antipeptone.
From these statements it is evident that a given proteid exposed to pepsin-proteolysis may give rise to a largenumber of products; in fact, to a far larger number than is implied by the names in the above scheme. Thus, at first glance you would be inclined to say there can be only three deuteroalbumoses, for example; one, a pure antibody, the other two, amphoalbumoses, differing from each other simply in their content of hemi- and anti-groups. It must be remembered, however, that the inner constitution of these bodies, as implied by the relative proportion of the above groups, may vary to almost any extent. Thus, every variation in the number of anti-groups split off from the original albumin molecule to form antialbumid means just so much of a change in the relative proportion of hemi- and anti-groups entering into the structure of both primary and secondary albumoses. Hence, as you can see, digestive proteolysis, even in gastric digestion, is a somewhat complex process. We have to deal not only with a number of bodies superficially unlike, as the primary and secondary proteoses and peptones, but these bodies may show marked variations in structure dependent upon the exact conditions attending their formation.
Evidently, the complexities attending digestive proteolysis are connected primarily with the complex nature of the proteids themselves, while proteolysis, as a process, is made possible through the natural tendency of the proteids to undergo hydration and cleavage.
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, butCahn94has 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 byMaly95many 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,96for 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.97This 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,98but 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;99it 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 whichthe 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 10 c.c. of fluid, was employed as the proteid material. In order to completely saturate the proteid contained in 20 c.c. of this neutral albumen solution, 50 c.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 + 50 c.c. 0.2 per cent. HCl + 30 c.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 + 25 c.c. 0.2 per cent. HCl + 30 c.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 20 c.c. of original albumen solution contained 1.6338 grammes of dry coagulable albumin. At the end of the forty-four hours,Acontained only 0.5430 gramme of unaltered albumin, or acid-albumin, whileBcontained 1.2225 grammes. That is to say, in the mixtureA, 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. InB, on the other hand, where the albumin was only half saturated with acid, 0.4113 gramme of the proteid was convertedinto 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 10 c.c. Two mixtures were prepared as follows:
A.Ten c.c. of the neutral albumen solution + 21.7 c.c. 0.2 per cent. HCl, the amount needed to completely saturate the proteid, + 40 c.c. of a weak solution of pepsin, perfectly neutral.
B.Ten c.c. of the albumen solution + 10.9 c.c. 0.2 per cent. HCl + 40 c.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 inAonly 0.1638 gramme of the proteid was undigested, while inB, 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 otherworkers101in 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 whichGillespie102is 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 beingperhaps 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’s103experiments, 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.104Hence, 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.105In 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: 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 11.30A.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 6,11.30A.M."2.15P.M.4.5c.c.,0.2per cent.HCl."5.00P.M.1.0""""February 7,8.45A.M.3.0"""""2.00P.M.1.0"""""5.00P.M.1.5""""February 8,8.30A.M.1.0"""""2.30P.M.0.0""""February 9,8.30A.M.3.0""""February 10,9.30A.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 nativeproteid, 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.106However 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 byRichet107quite 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,108however, assumed that solutions of leucin hydrochloride with pepsin would not digest albumin, but Salkowski andKumagawa109have 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;110i. e., digestion may be less rapid, especially on egg-albumin, a conclusion whichSalkowski111has 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 forthe 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. AsLangermann112has 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,Blum113has 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 purealbumin, 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.
While I am by no means willing to agree withBunge114that 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. AsCohn115has 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 notethat 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 itsmaximumaction 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 hasalready 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 theproportionof 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.