As a result of their experiments they suggested that in estimating. J3 Physiol. (I948) I07, I 6I2. 32I

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1 365 J3 Physiol. (I948) I07, I 6I2. 32I THE PRESENCE OF A PEPTIC SYNERGIST IN GASTRIC JUICE: ITS IMPORTANCE IN THE ESTIMATION OF THE PROTEOLYTIC ACTIVITY OF GASTRIC JUICE BY J. N. HUNT From Guy's Hospital Medical School, London, S.E. 1 (Received 7 November 1947) In 1945, Bucher, Grossman & Ivy described experiments which they interpreted as demonstrating the presence, in human gastric juice, of a pepsininhibitor/pepsin complex, which was dissociated by dilution. Their estimations of peptic activity were made with a modification of the Anson & Mirsky (1932) method. This method uses the increase in phenolic hydroxyl groups soluble in trichloroacetic acid and reacting with Folin & Ciocalteu's (1927) phenol reagent, as a quantitative index of the digestion of a substrate of carboxyhaemoglobin. As a result of their experiments they suggested that in estimating the peptic activity of human gastric juice it was necessary first to dilute the juice a hundredfold in order to eliminate the effect of the inhibitor. This point was obviously of some significance since it invalidated all estimations of the peptic activity of gastric juice in which this requirement had not been satisfied. This paper describes unsuccessful attempts to confirm their work and provides a possible explanation of the discrepancy by the demonstration of a peptic synergist in gastric juice. METHODS The proteolytic activity was estimated by a modification of the Anson & Mirsky method (Hunt, 1948) in which the carboxyhaemoglobin substrate is replaced by plasma proteins. It appeared from a priori consideration that if the product of the concentration of the gastric juice and the time of digestion were kept constant, in the absence of an inhibitor, the degree of colour developed, corresponding to phenolic hydroxyl groups, would be constant. On the other hand, if an inhibitor were present, a diluted sample with a correspondingly increased digestion time should be more active than an undiluted sample, owing to the dissociation of the inhibitorpepsin complex. RESULTS Twelve fresh 'resting juices' and seven fresh post-prandial juices were obtained from students. Each juice, diluted with an equal quantity of a solution of HCI (ph 2-1), was allowed to act on the substrate for 10 min. at

2 366 J. N. HUNT 350 C. and the colour corresponding to the increase in reactive phenolic hydroxyl groups was determined with Folin & Ciocalteu's reagent in a photoelectric colorimeter. It had previously been shown that the buffer capacity of the substrate was such as to make the estimation of the proteolytic activity of the gastric juice independent of its acidity, provided its ph lay between 1*6 and 4-1 (Hunt, 1948). Samples of the same gastric juices, diluted 1 in 20 with the same solution of HCI, were then allowed to act on the substrate for 100 min. and the colour was similarly measured. A total of 54 pairs of such determinations was made. The mean difference between the colour produced at the high dilution and low dilution was %, standard error of the mean %. The findings were the same with substrates of plasma, serum or boiled serum. These results appeared to provide a good reason for believing that the diluted juice did not in fact have any enhanced activity. Bucher et al. reported that samples of commercial and pure pepsin did not show the removal of an inhibiting effect when diluted. In this study similar results have been obtained for samples of crude commercial pepsin and for a highly purified specimen which gave a u.s.p. value of 1 in 45,000. In calculating their results, Bucher et al. used a standard curve relating proteolytic activity to colour developed in the reaction. This curve was produced experimentally from solutions of pepsin, not gastric juice, so that their results really expressed the 'pepsin-equivalents' of the samples examined. The proteolytic activity of gastric juice may only be legitimately expressed in 'pepsin-equivalents' if the curves for pepsin solution and gastric juice are identical. Therefore curves relating proteolytic activity to the colour difference between control and test were prepared for gastric juice and for commercial pepsin. These curves are shown in Fig. 1. They have been superimposed at A, thus arbitrarily making a solution of commercial pepsin (1.5 g./100 ml.) equivalent to a concentration of 158 pepsin units (Hunt, 1b48). It may be seen that the curves are not identical. Further, the discrepancy is such as to produce an error in reading the proteolytic activity of gastric juice from the curve for pepsin which might be interpreted as the removal of an inhibitor with dilution. For example, a gastric juice having an activity of 158 pepsin units/ml. would, after dilution with an equal volume of a solution of HCI, give an optical density of 0-64 on estimation. If this colour is used to read the activity of the gastric juice from the curve for pepsin, instead of the curve for gastric juice, it corresponds to 89 pepsin units instead of the expected value of half 158, namely 79, i.e. 50% dilutions would appear to remove inhibitor to the equivalent of 10 units. Bucher et al. reported that the amount of inhibition varied from one gastric juice to another; it is therefore relevant to inquire if the curve given in Fig. 1 for gastric juice differs significantly from that for commercial pepsin and whether the curve is sufficiently constant from one juice to another to allow it

3 PROTEOLYTIC ACTIVITY OF GASTRIC JUICE 367 to be used as a standard. The curve given in Fig. 1 for gastric juice was the mean curve for 138 points obtained from ten 'resting juices' and two obtained after injection of histamine. The constancy of this curve was tested with eight different samples of human gastric juice. The colours developed at various dilutions were determined, and the equivalent proteolytic activity was obtained from the standard curve for gastric juice. This directly obtained value luil a I I A. a x LU " 60-._ - 50 o // ~'8 /a /S //s le le le Curve for gastric juice Curve for commercial pel)siln Fig Pepsin unit/ml Liv '0 I I I g. pepsin/100 ml. Curves relating the proteolytic activity of gastric juice and commercial pepsin to the colour difference between control and test. was then compared with the theoretical activity calculated from the known degree of dilution and the activity of the original juice. The results of twentyseven duplicate estimations are shown in Table 1. The mean percentage difference between the two sets of readings is - 1'178 whilst the standard error of a single difference is 3-15 %. Since these experiments involved estimations at very low concentrations of gastric juice, without increasing the incubation time as would be done in practice, the agreement may be considered satisfactory.

4 368 J. N. HUNT TABLE 1. Variation of the relationship between peptic activity of gastric juice and the difference in colour between the control and test with different gastric juices Gastric juice Subject conc. (%) A * B 50.0 C *0 D 50.0 E E F G Colour difference between control and test (E x 100) * * *1 58* Peptic activity (units/ml) * * *8 10* * * *5 Peptic activity calculated from value obtained at 50% dilution (units/ml.) * * * *3 67* *8 21*9 16* * * TABLE 2. The error of the curve relating peptic activity to colour produced for gastric juice when used for estimating commercial pepsin Equivalent from curve Pepsin. (g./100 ml.) Colour difference relating peptic activity between control of gastric juice to and test colour (E x 100) (pepsin units/ml.) * * Expected value by calculation (units/ml.) * * Difference (%) - 2* '2 0-1* Difference (%) - 9* If the values obtained with the commercial pepsin are similarly examined it may be seen in Table 2 that at all concentrations above 025 g. pepsin/100 ml. there is a considerable difference between observed and calculated values. Since the differences are at least three times the mean standard error of the curve for gastric juice as assessed from Table 1, it appears likely that the curves for gastric juice and commercial pepsin are different. Lest the discrepancy

5 PROTEOLYTIC ACTIVITY OF GASTRIC JUICE 369 between the curves should be considered as due to a chance variation in substrate or commercial pepsin, a few additional points have been obtained for another commercial pepsin with a second sample of substrate. All the experimental points obtained for commercial pepsin have been included in Fig. 1. Preliminary experiments have been made to investigate this difference between the course of proteolysis with human gastric juice and commercial pepsin. If the gastric juice contained an accessory substance which assisted the pepsin to digest the substrate more effectively, mixing samples of gastric juice and pepsin should give a higher activity than the mean of the samples estimated separately. Two samples of gastric juice when mixed in equal proportions gave a colour which corresponded to the mean of their activities as measured from the standard curve for gastric juice. TABLLE 3. The synergic effect of estimating the proteolytic activity of gastric juice and pepsin together Difference in Peptic activity colour between from curve for control and test Mean gastric juice Enzyme source (E x 100) (E x 100) (pepsin units/ml.) Gastric juice A * Purified pepsin about mg./100 ml *0) Equal parts of pepsin 50-2 solution and gastric 50-2 juice ' ml. of enzyme solution was used in each estimation. Table 3 shows results typical of six experiments in which a solution of purified pepsin (1 in 45,000) and a human gastric juice were estimated separately and when mixed in,equal parts. It may be seen that the colour produced by the mixture is greater than that produced by either of the two solutions separately. If the curve relating activity to colour were the same for pepsin and gastric juice the ex'pected optical density produced by the mixture would be about The difference between this and the colour actually obtained corresponds to an optical density of 0*075, which is equivalent to 10 pepsin units on this part of the curve. Since the standard error of an individual estimation by the method employed is 2-6% (Hunt, 1948) the difference is six times the standard error of a triplicate estimation, so that there can be no doubt of its significance. This enhancing effect of gastric juice upon pepsin was not shown when gastric juice or a solution of purified pepsin, previously heated to for 20 min. and cooled, was added to a solution of pepsin. The heated solution of pepsin when containing only about 15 mg./100 ml. had a marked

6 370 J. N. HUNT inhibiting action both on gastric juice and pepsin. These results are compatible with the presence of a synergic enzyme in the gastric juice but do not, however, exclude other possibilities. DISCUSSION The difference between the curves relating proteolytic activity to the colour developed for commercial pepsin and gastric juice provides a reasonable explanation of the failure to demonstrate the inhibiting complex postulated by Bucher et al. There are, however, other possibilities. It might be suggested that plasma proteins are a special substrate upon which the effect of diluting gastric juice is not apparent. If this is the case, it is an advantage in the use of this substrate, but one which, according to the results of Ihre (1938), it shares with casein. It must be admitted that although the difference between the two curves accounts qualitatively for the effect described by Bucher et al., it does not account for the twofold enhancement described by these workers. It seems possible, therefore, that the two curves may be even less coincident with the more dilute substrate of carboxyhaemoglobin employed in their work. The results of the estimation of mixtures of pepsin and gastric juice are compatible with the hypothesis that the observed enhancement is due to a second enzyme in gastric juice which converts the substrate into a form more readily digested by pepsin. When pepsin and gastric juice are mixed, the added pepsin acts on an altered substrate and shows enhanced activity. Since Northrop (1939) has already shown the presence of gelatinase in gastric juice, there is nothing improbable in this suggestion. When the first experiments were made to establish the relationship between the proteolytic activity of gastric juice and the difference in colour between the control and test, a stored sample of gastric juice was used. Subsequent comparison with curves made from fresh samples showed that the stored juice gave an increased rate of digestion at low concentrations as compared with fresh juice. This observation may perhaps be coupled with the well-known finding that pepsin solutions are unstableswhilst gastric juice shows remarkable stability in its proteolytic powers. This relative stability of proteolytic activity of gastric juice might be due to a gradually increasing proportion of the substrate made available to a second more active enzyme as the pepsin became progressively inactivated. The hypothesis that the difference observed between gastric juice and commercial pepsin is due to a synergic enzyme receives support from the work of Kraut & Tria (1937) who, using a method originally described by Briicke in 1875, prepared a proteolytic enzyme from gastric mucosa which was quite distinct from pepsin as prepared by Northrop. Their enzyme acted synergically with pepsin and had an initial reaction rate greater than pepsin, two properties which have been demonstrated for gastric juice. It appears, therefore, that their enzyme may be present in gastric juice as well as in gastric mucosa.

7 PROTEOLYTIC ACTIVITY OF GASTRIC JUICE 371 As a consequence of the difference between the curves for commercial pepsin and gastric juice, it appears that the relationship between the degree of proteolytic activity and the index substance measured in any method of estimating the proteolytic activity of gastric juice, should be established with gastric juice and not with pepsin. The proteolytic activity of gastric juice may only legitimately be expressed in terms of weights of pure pepsin if the concentration of a standard solution is defined. SUMMARY 1. Using a modification of the Anson & Mirsky method of estimating proteolytic activity, no evidence of a pepsin-inhibitor/pepsin complex dissociating on dilution has been found. 2. An hypothesis, supported by experiment, is put forward to account for the positive findings of previous workers. 3. The reproducibility of the curve relating peptic activity of gastric juice to the concentration of the products of digestion is delimited for eight gastric juices. 4. The difference between this relationship for commercial pepsin and human gastric juice is shown. 5. Preliminary experiments have given results compatible with the presence of synergic enzyme in gastric juice. 6. The significance of this finding as it relates to methods of estimating the proteolytic activity of gastric juice is discussed. I am indebted to Messrs Parke Davis & Company for a supply of purified pepsin. REFERENCES Anson, M. L. & Mirsky, A. E. (1932). J. gen. Physiol. 16, 59. Brucke, E. V. (1875). Vorles. u. Physiol. 2. Aufl., 1, 299. Bucher, G. L., Grossman, M. I. & Ivy, A. C. (1945). Gastroenterol. 5, 501. Folin, D. & Ciocalteu, V. (1927). J. biol. Chem. 73, 627. Hunt, J. N. (1948). Biochem. J. 42, 104. Ihre, B. J. E. (1938). Acte med. Scand. Suppl. xcv. Kraut, H. & Tria, E. (1937). Biochem. Z. 290, 277. Northrop, J. H. (1939). CrystaUine Enzymes. New York: Columbia University Press.