factors. directly. There are many, however, who regard the hydrochloric acid degree of stimulation of the stomach cells [Roseman, 1927; Katsch &

Transcription

1 308 J. Physiol. (I940) 97, 308-3I5 6I ON THE PRIMARY ACIDITY OF THE GASTRIC JUICE BY TORSTEN TEORELL From the Department of Medical Chemistry, University of Uppsala, Sweden (Received 10 July 1939) As early as 1898 Pavlov advanced the theory that the gastric hydrochloric acid is secreted from the mucosa glands at a high and constant concentration, independent of other conditions such as secretion rate, etc. This so-called " primary acidity " undergoes secondarily a reduction to lower and variable values, owing to interference from various acidity regulating factors. Most workers on gastric physiology have adopted Pavlov's view, although so far nobody has been able to examine the primary juice directly. There are many, however, who regard the hydrochloric acid concentration of the "primary juice" as being variable according to the degree of stimulation of the stomach cells [Roseman, 1927; Katsch & Kalk, 1926; and others]. Hollander [1932], in particular, has defended the Pavlov thesis and has attempted to accumulate indirect evidence in its favour. He claims that the primary acidity corresponds to blood isotonicity (ca. 167 mn.'). His evidence is based on inferences from the observed correlation between acidity and neutral chloride of gastric juice samples. Hollander's method of arguing is purely mathematical and involves an extrapolation to the acidity value where the neutral chloride is zero. The fact that this extrapolation leads to a constant figure of about 167 mn. does not necessarily prove that the primary acidity of samples containing neutral chlorides is also 167. A similar mathematical procedure employed by Liu, Yuan & Lim [1934] is open to the same criticism. Although the Pavlov-Hollander hypothesis is attractive in many respects, from a critical standpoint one must conclude that it lacks a convincing foundation. The object of the present study was to attempt a direct examination of the acidity of the primary juice. The mode of attack was based on new concepts as regards the mechanism of the acidity regulation [the 1 mn. = milli-normal = milli-equivalents per litre.

2 ON THE PRIMARY ACIDITY OF THE GASTRIC JUICE 309 "diffusion theory", Teorell, 1933, 1935, 1939a; cf. also Engestrom, 1935; lhre, 1938]. Remarks on the acidity regulation.' Regardless of their views on the composition of the primary juice, all workers in this field agree that there exist secondary acidity-reducing factors. The nature of these factors is still a matter of controversy. Pavlov [1898] and many later investigators consider a neutralization by gastric mucus as the most important factor. Others have suggested a neutralization process by bicarbonate or buffers of extragastric origin (Boldyreff's [1911] duodenal regurgitation), or of intragastric origin [Hollander, 1938]. There are many who believe in a pure dilution by an intragastric "Verdiinnungssekretion" [McLean, 1928; Katsch and Kalk, 1926; and others]. Working with gastric juice secretion of cats, induced by histamine stimulation, the writer [1933] was unable to find indications of neutralizing or diluting processes. Instead he was forced to adopt a purely physico-chemical view and advanced a "diffusion theory" as the only satisfactory explanation of the experimental findings. The chief principle is the following: the hydrochloric acid diffuses steadily out of the stomach content into the mucosa cells or the blood, and is exchanged against alkali chlorides ("neutral chlorides") which diffuse the other way. Expressed technically, there is a continuous process of ionic interdiffusion or exchange diffusion going on in the stomach. (For details of this theory see Teorell [1933, 1939a].) Principle of experimental procedure. Experiments on the introduction of acids into cats' stomachs show that the loss of acidity is proportional to the prevailing hydrogen-ion concentration [Teorell, 1933, 1939 a], in accordance with the laws of diffusion and the behaviour in model experiments [Teorell, 1939 b]. From this it follows that there are two ways open for a prevention of secondary acidity reduction: (a) A lowering of the hydrogen-ion concentration of the stomach content by dilution of the acid secretion. This method has been utilized by Engestrom, who received the HC1 secretion in a large volume of water introduced into the stomach. A drawback, however, is that very large volumes of diluting solutions have to be employed in order to prevent HCI loss completely (for example, a reduction of the back diffusion rate to a tenth requires a diluting volume which is nine times larger than that of the acid secretion). (b) A lowering of the hydrogen-ion concentration by mixing the acid juice with a suitable buffer solution. Provided this has a sufficiently large acid-binding capacity, it is possible to use comparatively small volumes of the buffer. In either case one can determine the amount of HCI secreted by titrating the buffer and juice withdrawn with NaOH back to the ph of the original juice-free buffer. If the solution introduced is osmotically 1 The subject of primary acidity and acidity regulation has recently been reviewed by Hollander [1938]. Cf. also the dissertations by Ihre [1938], Engestrom [1935] and Teorell [1933].

3 310 T. TEORELL so adjusted that it does not undergo any volume changes in the stomach, it is obvious that any increase of volume is a direct measure of the volume of the secretion. Finally, by dividing the amount by the volume one obtains the concentration of HC1 in the secretion. The following numerical example illustrates the principle: 5 0 c.c. of an "isotonic" buffer (ph 5.9) were introduced into a just emptied but secreting stomach. After a certain time 6-5 c.c. could be withdrawn from the stomach. [The ph was now Without buffer the ph would probably have been about 1f0, hence there has been achieved a 56-fold reduction of the acidity ( =the logarithm of 56).] The sample withdrawn required 2-8 c.c. N/10 NaOH for adjustment of the ph back to 5*9, i.e. the amount of HC1 secreted corresponds to 0*28 mm. The volume increment was = 1 5 c.c., hence the primary acidity of the secretion is equal to 0281x1000= 1*5 186 mn. METHODS After several preliminary trials a suitable buffer substance was found in glycocol. In a M solution it is subject to insignificant volume changes in the stomach. The substance is very poorly permeable through the mucosa (cf. Teorell [1939 a], and the total nitrogencolumn of Table II), therefore, its buffering capacity remains practically constant. The poor permeability probably explains why one has to employ solutions hypotonic to the blood in order to avoid osmotic volume changes (a 0*2 M solution has a freezing point of compared with 0.60 of the cat's blood). At 6-0, the ph of the plain glycocol solution, the buffering capacity is quite small, therefore a sharp end-point for the back titration is obtained (see Exp. 1 (b)). The ph measurements and NaOH titrations were performed by aid of a glass electrode arrangement. The chloride titrations were carried out electrometrically. Nitrogen determinations were made on aliquot parts, as described by Teorell [1928]. The volumes were measured directly, the accuracy being ± 0-2 c.c. The treatment of the experimental animals was the same as described in detail elsewhere [Teorell, 1939 a]. The mode of calculation is illustrated above and by Exps. 1 and 2. RESULTS Prior to the main experiments some controls were performed in order to test the reliability of the methods employed. The results are quoted as Exps. 1-4.

4 ON THE PRIMARY ACIDITY OF THE GASTRIC JUICE 311 Experiment 1. Titration controls in vitro. (a) 5-0 c.c. of a 0-9 % NaCl+2-0 cc. of N HCl were titrated with N/l0 NaOH. End-point ph 7-0, total NaOH consumption 3-54 c.c. Volume "increment" 2-0 c.c., hence the "primary acidity" is equal to 2-0 x 1000=177 mn. (calc. 177). (b) 5-0 c.c. of 0-2 M glycocol c.c. of a N HCI titrated with N/10 NaOH. End-point ph 6-0. c.c. NaOH... Initial ph NaOH consumption 3-55 c.c. Volume " increment" 2-0 c.c., hence the "primary" acidity is equal to x 1000 = 178 mn. (calc. 177). 2-0 N/10 AgNO3 consumption 3-56 c.c. (calc. 3-54). Exp. 2. Recovery controls in cat's stomach. Glycocol + HCl mixtures (a slight fasting secretion was going on). (a) 5-0 c.c. of a solution (5-0 c.c. 0-2 M glycocol +2-0 c.c N HCI) were introduced into the emptied stomach (at p.m.). At a volume of 5-4 c.c. was recovered (ph 2-54) which consumed 3-40 c.c. N/10 NaOH (end-point ph 5-5-6). The N/10 AgNO3 consumption of the total sample was 4-20 c.c. The theoretical NaOH consumption corresponding to the amount of HCl introduced ought to be - x = 2-53 c.c. This figure is lower than the figure actually determined, 7. indicating the presence of an acid secretion corresponding to =0-87 c.c. N/10 NaOH. If one postulates the primary acidity to be N, the secreted volume is calculated to be (0-1 x 0-87) =0-50 c.c. Now, the actual volume,increment was =0-4 c.c., hence there is a deficit of 0-1 c.c. If it is assumed that all hydrogen ions were secreted together with chloride ions, one can calculate the "neutral" chloride concentration of the stomach content to be ( ) x 100 = 15 mn. 5.4 (b) The foregoing procedure was repeated: 5-0 c.c. fresh glycocol + HCI mixture were introduced at p.m., 5-0 c.c. were recovered at (ph 3-48). NaOH consumption 2-95 c.c., AgNO3 consumption 3-57 c.c. Acid increment corresponding to =0-42 c.c. NaOH or 0-24 c.c. primary acid. The actual increment was 0, hence a volume deficit of 0-24 c.c. The neutral chloride concentration was (3 2-) x 100 = 12 mn. 5-0 Exp. 3. Recovery controls in cat's stomach. Glycocol alone (a slight fasting secretion was still going on). (a) 5-0 c.c. of 0-2 M glycocol were introduced into the emptied stomach at 1.10 p.m., 5-0 c.c. (ph 3-52) were recovered at NaOH consumption 0-55 c.c. corresponding to 0-31 c.c. of primary acid, hence a deficit of 0-31 c.c., AgNO3 consumption 1-17 c.c., neutral chloride concentration equal to 12 mn. Total nitrogen of the sample was 13-8 mg. (calc. 14-0). (b) Foregoing repeated c.c. recovered (ph 3-66) NaOH consumption 0-50 c.c., AgNO3 consumption 1-08 c.c., yielding a volume deficit of 0-29 c.c. and a neutral chloride value of 12 mn. Total nitrogen of the sample 13-8 mg. (calc. 14-0).

5 312 T. TEORELL From the Exps. 1-4 one can infer that: (1) the titration methods are satisfactory; (2) there is a slight water "resorption" of c.c. during the time interval of 15 min.; (3) there occurs an increase of the neutral chloride level up to about 12 mm. during 15 min.; (4) the glycocol does not diffuse out of the stomach (Nis constant). The main experiment is recorded in Table I. When calculating the primary acidity the volume was corrected with +0-2 c.c. to account for the water resorption. TABLE I. Exp. 4. Gastric juice secretion in the presence of glycocol buffer. The same cat as in Exp. 3. Every 15 min. total emptying of stomach content and 5-0 c.c. fresh glycocol solution introduced. At 1.48 p.m. subcutaneous injection of 0-6 mg. histamine hydrochloride. * ~~~~~~~~~~Concentration Total Volume Volume N of Total Neutral H* Primary re- in- sample H* Cl Cl Total acidityt Time covered crement ph mg. MN. inn. mn. Cl mn (5-0 c.c. introduced) 5-46 (14-0) * H denotes titrable acidity to ph t 0-2 c.c. has been added to the volume increment to allow for water resorption. + Calculated value. The results of Exp. 4 (Table I) should be compared with those of Exp. 5 (Table II) which is a standard gastric juice experiment where "secondary" acidity only was determined. TABLE II. Exp. 5. Gastric juice production without extragastric admixture. (The same cat as in Exps Every 15 min. total emptying of the stomach. At 8.20 p.m. subcutaneous injection of 0-6 mg. histamine hydrochloride.) Volume Total N Total Total Neutral Time recovered of sample acidity* chloride chloride p.m. c.c. mg. inn. mn. inn * End-point ph 7-4.

6 ON THE PRIMARY ACIDITY7 OF THE GASTRIC JUICE 313 A comparison between Tables I and II shows the following: (1) The primary acidity lies on a considerably higher level than the total acidity, and appears to be independent of the rate of secretion. There is a considerable fluctuation among the figures obtained, between mn. It should be remembered, however, that these figures are very sensitive to errors in the volume determinations. A deficit within the limits of error, 0-2 c.c., causes a 10 % increase of the primary acidity when the volume increment is 2 c.c. and still more when the increments are smaller. It may therefore be justified to disregard all samples with less than 2 c.c. increments, in which case the following figures only can be accepted as reliable mn. A similar experiment on another cat yielded for four different samples mn. The average of these eight figures for the primary acidity is mn. (2) The neutral chloride level in the glycocol experiments is somewhat fluctuating. The neutral chloride level in the standard experiment is considerably higher. The reason for this behaviour will be considered in the discussion. (3) There is no increased loss of glycocol due to diffusion during actual gastric juice secretion. This is shown by the nitrogen figures. DIsCUSSION The main error in the method employed rests in the difficulty of obtaining accurate volume figures. Dilution measurements with the aid of a fixed " reference substance " (phenol red, etc.) would perhaps improve the results. Owing to this circumstance one can only ascribe significance to figures obtained at the higher secretion rates. It can also be objected that the back titration with NaOH over the ph range includes not only the HCl+glycocol-HCl, but also other base binding substances which might appear in the stomach contents. It can likewise be suggested that some HCI can be neutralized by other substances than the buffer, having an acid binding power beyond ph Of these two opposing possibilities the former is the most probable one, as indicated by the very small neutral chloride figures recorded in Table I. Hence it is likely that we have been dealing with NaOH titration values which are slightly too high; these in turn have given rise to primary acidity figures which are somewhat too high. The average value of the primary acidity so obtained, mn.,

7 314 T. TEORELL would even after due allowance for a possible "extra" base binding, remain higher than the figure , indirectly arrived at by Hollander [1932]. It would agree rather with the suggestions by Liu, Yuan & Lim [1934] that the parietal secretion is hypertonic with respect to blood. However, the total chloride values of the acid juice from cats are in general higher than in dog or human, values above 180 mm. being not uncommon (cf. Table II). Therefore, the matter of the possible isotonicity of the primary juice with respect to the blood cannot be decided upon until freezing-point determinations or appropriate calculations of the osmotic pressures have been performed. In several communications it has been shown that there is an inward diffusion of alkali chlorides from the mucosa or blood to the stomach contents [Teorell, 1933, 1939 a], and it is therefore most natural to explain the excess chloride over the "acid" chloride, i.e. the neutral chloride, as a product of such an inward diffusion. The results obtained here may allow some speculations as to the position of the mucosa elements in which the back diffusion of hydrochloric acid takes place. It is generally assumed that the parietal secretion enters the stomach cavity via the " crypts " of the mucosa glands. As the crypts are microscopically narrow, it seems likely that the outwardly directed stream of secretion prevents the buffer from entering them. As nevertheless the figure of primary acidity was found to be high and, so far as could be judged, independent of the secretion rate, it might be inferred that no acidity reduction occurred during the passage through the crypts. The evidence is not conclusive; on the contrary, there are some signs which may indicate that some "intraglandular acidity reduction" [Engestr6m, 1935] has occurred, at least at the lowest secretion rates. These signs are the decreasing ratios (titrable acidity/ total chloride) of Table I. On the whole, however, it seems probable that the main acidity reduction takes place on the parts of the mucosa which are directly exposed to the stomach cavity. Finally, it may be emphasized, that the observation that a buffer inhibits the reduction of the titrable acidity can be taken as a direct support for the view that a back diffusion process of the H ions of the secreted " primary " HCI is a dominating mechanism in " regulating " the gastric juice acidity. SUMMARY 1. It has been pointed out that indirect evidence only is available in favour of the Pavlov hypothesis regarding the "primary" acidity of the gastric juice.

8 ON THE PRIMARY ACIDITY OF THE GASTRIC JUICE A new method is outlined which allows a direct determination of the primary acidity. The principle is based on observations that the rate of acidity reduction is proportional to the hydrogen-ion concentration of the stomach contents (in accordance with the "diffusion theory"). The essential part of the technique is the utilizing of the acid buffering power of glycocol. 3. On cats, stimulated with histamine, reliable values of the primary acidity were obtained only at high rates of secretion. The average value was mn., but there are reasons for believing that this value is slightly too high. 4. The possibilities of an "intraglandular" acidity regulation have been discussed. REFERENCES Boldyreff, W. [1911]. Ergebn. Physiol. 11, 121. Engestrom, T. [1935]. Acta Med. Scand. supplem. LXVI. Hollander, F. [1932]. J. biol. Chem. 97, 585. Hollander, F. [1938]. Review of work in Amer. J. Dig. Dis. 5, 364. lhre, B. [1938]. Acta Med. Scand. supplem. xcv. Katsch, G. & Kalk, H. [1926]. Klin. Wschr. p Liu, Yuan, & Lim [1934]. Chinese J. Physiol. 8, 1. McLean, H. [1928]. Modern Views on Gastric Disease. London. Pavlov, I. P. [1898]. Die Arbeit der Verdauungsdriisen. Wiesbaden. Roseman, R. [1927]. Handb. norm. path. Physiol. 3, 819. Teorell, T. [1928]. Acta Med. Scand. 68, 305 (N-method). Teorell, T. [1933]. Skand. Arch. Physiot. 66, 225. Teorell, T. [1935]. Acta Med. Scand. 85, 518. Teorell, T. [1939 a]. J. Gen. Physiol. 23, 263. Teorell, T. [1939 b]. Unpublished observations.