A RADIAL DIFFUSION ASSAY FOR PEPSINOGEN AND PEPSIN

Transcription

1 GASTROENTEROLOGY Copyright 1969 by The Williams & Wilkins Co. Vol. 56, No.1 Printed in U.S.A. A RADIAL DIFFUSION ASSAY FOR PEPSINOGEN AND PEPSIN I. MICHAEL SAMLOFF, M.D., AND MARTIN S. KLEINMAN, M.D. Gastroenterology Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York A radial diffusion assay for the determination of gastric pepsin and uropepsin is developed. Urine, gastric juice, or solutions of hog pepsinogen are allowed to diffuse radially from circular wells in agar gel which contains acid-hemoglobin. After a period of incubation, the undigested hemoglobin is fixed with acid-alcohol, and after drying, the agar is stained for protein. In the presence of peptic activity, a circular unstained zone is seen whose diameter is linearly related to the logarithm of the concentration of enzyme. In previous reports from this and other laboratories, a method for the separation and identification of pepsinogens by electrophoresis of extracts of gastric mucosa in agar gel has been described. I. 2 In this method, the agar gel is incubated in acidhemoglobin or acid-albumin after electrophoresis and then stained for protein. Proteolytic activity is indicated by an unstained zone since the products of digestion do not react with specific protein stains. It occurred to us that a quantitative assay for pepsinogen and pepsin might be developed by allowing the test solution to diffuse radially from a well in agar gel which contains substrate (acid-hemoglobin). This report presents the details of the method and demonstrates that it is applicable to the assay of pepsinogen and pepsin from a number of different sources. Materials and Methods Agar gel. The agar gel was prepared by adding 2.8 g of Noble agar (Difco Laboratories, Received April 11, Accepted June 20, Address requests for reprints to: Dr. I. Michael Samloff, Department of Medicine, Harbor General Hospital, 1000 West Carson St., Torrance, C;Uifornia This investigation was supported by Grant AM from the National Institutes of Health, United States Public Health Service. Dr. Kleinman was supported by training Grant TO 1 AM 05177, also from the National Institutes of Health. 30 Detroit, Mich.) to 100 ml of distilled water containing 10 mg of sodium azide and heating the suspension on a hot plate-magnetic stirrer. The agar gel may be used immediately or stored in tightly stoppered tubes at 4 C where it is stable for several months. Ionagar no. 2 (Consolidated Laboratories, Chicago Heights, Ill.) gave similar results, but Agarose (Mann Research Laboratories, New York, N. Y.) was not satisfactory because of poor staining characteristics. Hemoglobin. Bovine hemoglobin type II (Sigma Chemical Company, St. Louis, Mo.) was dissolved in distilled water to a concentration of 1.2 g per 100 ml. Hemoglobin from other sources gave less satisfactory results. Buffer. An acetate-hydrochloric acid buffer was prepared by dissolving 16.4 g of anhydrous sodium acetate in 260 ml of 1.0 N HCl and diluting the solution to 1.0 liter with distilled water. Preparation of agar-substrate mixture. The required amount of agar-gel was melted in a boiling water bath and then placed in a water bath at 60 C. For each 9.0 ml of agar, 1.0 ml of hemoglobin and 2.0 ml of buffer were mixed and added slowly to the melted agar. The contents of the tube were stirred to disperse the substrate evenly throughout the agar. The final concentrations of agar and hemoglobin were 2.1 g per 100 ml and 0.1 g per 100 ml, respectively. The ph, as estimated by adding 9.0 ml of distilled water to the acid-hemoglobin, was 2.0. Preparation of slides. Glass microscope slides were covered with the above mixture to a uniform thickness of 1 mm. In this study,

2 January 1969 RADIAL DIFFUSION ASSA Y FOR PEPSINOGEN AND PEPSIN 31 LKB (Rockville, Md.) immunodiffusion equipment was used for the preparation of the slides. After the gel had set, four 3-mm diameter wells were punched in each slide with LKB cutter 6865-A. At this stage, the slides may be used immediately or stored in a moist chamber at 4 C, where they are stable for at least 2 weeks. Radial diffusion assay. The agar-coated slides, if stored in the cold, were allowed to return to room temperature. Then the wells, which hold approximately 8 Illiters, were filled with urine, gastric juice, or a solution of hog pepsinogen or pepsin, care being taken to fill the wells equally. A small glass capillary tube (Will Scientific, Inc., no ) was used for this purpose. The slides were then placed in a humid chamber, and this was placed in an incubator at 37 C. Incubation times of 2V2 hr for urine and 1 hr for gastric juice were selected for routine use. Following incubation, the slides were immersed in 5% acetic acid in 50% ethanol overnight to stop the reaction and to precipitate undigested protein. After drying, the slides were stained with amido black. Proteolytic activity is indicated by the presence of a circular clear zone around the well (fig. 1). Since this represents the interaction of enzyme and substrate, it is referred to as the reaction zone. The slides were enlarged 4-fold with a conventional photo-enlarger, and at least two diameters of the projected circles were measured to the nearest 0.5 mm. Whenever possible, slides were selected for measurement at random to minimize observer bias. The values given in the text are for the net diameter of the reaction zone (total diameter less diameter ofwell/4). Uropepsin assay. Uropepsin was also determined by a modification of the Anson and Mirsky3 procedure in order to correlate the results obtained by the conventional method with the radial diffusion assay. Duplicate sample and control tubes were set up for each urine sample. Each tube received 4.0 ml of hemoglobin and 1.0 ml of 0.3 N HCl. Then 0.5 ml of urine was placed in each of the 2 sample tubes, and after mixing the sample and control tubes were covered and incubated in a water bath for 1 hr at 37 C. The reaction was stopped with 9.0 ml of 0.3 N trichloracetic acid. The control tubes received in quick succession 0.5 ml of urine and 9.0 ml of trichloracetic acid. The tubes were then centrifuged, and the amount of tyrosine-like substances liberated from the hemoglobin substrate was measured in 1.0 ml of the supernatant by the Folin Ciocalteu method. The difference between the FIG. 1. Radial diffusion assay. Incubation for 2Y2 hr at 37 C. The wells in the top slide contained, from left to right, hog pepsinogen 1, 4, 16, and 64 Ilg per ml. Samples of urine were placed in the eight wells in the lower two slides. To convert diameter of reaction zone to proteolytic activity, a standard curve is plotted for the hog pepsinogen standards, and the uropepsin activity is expressed as micrograms equivalent hog pepsinogen per milliliter. mean optical density for the two sample tubes and the two control tubes was used to calculate the result. A standard curve was constructed by the use of incubated duplicate standard tubes containing, instead of urine, 250 Ilg and 500 Ilg of tyrosine, respectively. Results Quantitative relations. Twelve concentrations of crystalline hog pepsinogen (Worthington Biochemicals, Inc., lot no. PG 6LC) from 128 to Ilg per ml were prepared in M acetate buffer, ph 5.6, and examined for proteolytic activity by the radial diffusion assay. The relationship between the logarithm of enzyme concentration and the diameter of the reaction zone was essentially linear over the entire range of pepsinogen con-

3 32 SAMLOFF AND KLEINMAN Vol. 56, No.1 centration (fig. 2). Similar results were obtained with hog pepsin. Urine was serially diluted in 0.15 M saline, and each dilution was examined for proteolytic activity by the radial diffusion assay. The diameter of the reaction zone was found to be linearly related to logarithm of urine concentration (fig. 3). Ten samples of urine were examined for proteolytic activity by both the modified Anson-Mirsky and the radial diffusion assays (fig. 4). A high degree of correlation was found between the diameter of the reaction zone and the logarithm of uropepsin concentration (r = 0.94). Reproducibility and variation due to the use of different slides and different po- sitions on a single slide. Three agar-coated slides were prepared with four wells on each slide. Each well received the same sample of urine. The results and statistical analysis of the data are presented in tables 1 and 2. These indicate that there ' UROPE PSIN [u/ml) FIG. 4. Relationship between diameter of reaction zone and logarithm of uropepsin concentration. Each dot represents the mean value of duplicate determinations of proteolytic activity in one urine by the radial diffusion assay and by the modified Anson and Mirsky assay '0 ' HOG PEPSINOGEN ["g/ml) FIG. 2. Relationship between diameter of reaction zone and hog pepsinogen concentration. This is a semi-logarithmic plot. The regression line in this and all subsequent figures was calculated by the method ofleast squares. TABLE 1. Analysis of the diameter of reaction zone as a function of different slides and different positions on a single slide Wen Slide no. Total I Total '--,': 6-4 -"'.32-':"6--"': " :-4----'UcoN=D IlUTED URINE DILUTION FIG. 3. Relationship between diameter of reaction zone and dilution of urine. This is a semi-logarithmic plot. TABLE 2. Analysis of variance Source of I Sums of I Degrees of I Variance variation squares freedom Variance ratio (F) ---- Slides. " Wells Error. " Total

4 January 1969 RADIAL DIFFUSION ASSA Y FOR PEPSINOGEN AND PEPSIN 33 was no significant variation in the diameter of the reaction zone among slides or among wells. The coefficient of variation using the total variance of all 12 observa: tions to calculate the standard deviation was 2.6% ofthe mean. ' zone to time of incubation. The growth in the diameter of the reaction zone was linearly related to the square root of time of incubation (fig. 5). With prolonged incubation, the margins of the reaction zone became increasingly indistinct, making it difficult to obtain accurate measurements. Incubation times of from 2 V2 to 4 hr for urine and from 1 to 2 hr for gastric juice were found to be satisfactory under conditions given above for gel strength and temperature of incubation. zone to temperature of incubation. Uropepsin activity was determined on slides incubated at 23 C or at 37 C for from 1 to 8 hr. There was no difference in the diameters of the reaction zones of replicate samples after incubation for 1 hr (fig. 5). This is attributed to a delay in the agar gel's reaching incubator temperature. After 1 hr the rate of increase of the diameter of the reaction zone at 37 C was approximately twice that at 23 C. 4 6 HOURS -- 23' FIG. 5. zone to time and to temperature of incubation. The hours of incubation (abscissa) are plotted on a square root scale. The rate of increase in the diameter of the reaction zones for urines A and B is linearly related to the square root of time and is approximately twice as great at 37 C than at 23 C. 6 ~." o HOG PEPSINOGENl,ug/ml UROPEPSIN 3.4 u/ml AGAR (gm/looml) FIG. 6. zone to concentration of agar gel. Incubation for 2 V2 hr at 37 C. zone to concentration of agar gel. Three concentrations of agar gel were prepared to give final concentrations of 2.1, 1.6, and 1.1 g per 100 ml. The diameters of the reaction zones produced by replicate samples of urine and of hog pepsinogen were determined after incubation for 2 V2 hr at 37 C. The diameters of the reaction zones were indirectly related to the concentration ofthe agar gel (fig. 6). Standards. To convert the diameter of the reaction zone produced by urine or gastric juice to proteolytic activity, a standard curve is constructed by incubating known concentrations of hog pepsinogen along with the test samples (figs. 1 and 2). The standards, which are stable for at least 1 week when stored at 4 C, are included with each assay. Proteolytic activity of the test samples is expressed as micrograms equivalent hog pepsinogen per milliliter. Discussion The hemoglobin method of Anson and Mirsky3 and modifications thereof have been widely used to determine the level of peptic activity in gastric juice and urine. The radial diffusion assay is merely another modification of this general method in which the extent of hemoglobin proteolysis is determined directly by measuring the diameter of the reaction zone rather than indirectly by measuring the amount of tyrosine-like substances released during the period of incubation.

5 34 SAMLOFF AND KLEINMAN Vol. 56, No.1 The radial diffusion assay is convenient, simple, and requires a minimum of technical ability and equipment. A singular advantage is the facility with which several hundred specimens can be examined daily. This has proven of value in gel filtration experiments of gastric mucosal extracts where it is of interest to determine which eluate fractions contain proteolytic activity. The diffusion assay also provides a possible means for a large scale survey of the population for chronic atrophic gastritis and gastric atrophy, conditions in which uropepsin activity is usually low or absent.4. 5 The hemoglobin method of Anson and Mirsky3 for gastric pepsin requires up to a 200-fold dilution of gastric juice. There are both theoretical and practical objections to this because dilution of gastric juice often results in an apparent increase of the total peptic activity of the sample. 6 The use of undiluted gastric juice in the radial diffusion assay overcomes these objections. The general method of radial diffusion is applicable to the estimation of several enzymes of interest to the gastroenterologist. Preliminary studies indicate that the levels of amylase in serum, urine, and duodenal juice, trypsin, chymotrypsin, and carboxypeptidase in duodenal juice, and carboxypeptidase in stool are readily estimated by incorporating suitable substrates for these enzymes in agar gel. REFERENCES 1. Kushner, I., w. Rapp, and P. Burtin Elec trophoretic and immunochemical demonstra tion of the existence of four human pepsino gens. J. Clin. Invest. 43: Samloff, I. M., E. V. Barnett, and M. D. Turner Immunization of rabbits with hog pep sinogen. I. Autoantibodies to pepsinogen and elevated serum pepsinogen levels. Gastroen terology 52: Anson, M. L., and A. E. Mirsky The esti mation of pepsin with hemoglobin. J. Gen. Physiol. 16: Farnsworth, E. B., H. Speer, and H. L. Alt The quantitative determination of a pepsin. like substance in the urine of normal individ uals and of patients with pernicious anemia. J. Lab. Clin. Med. 31: Segal, H. L., and I. M. Samloff Tubeless gastric analysis techniques in diagnosis of chronic gastritis. J. A. M. A. 197: Bucher, G. R., M. I. Grossman, and A. C. Ivy A pepsin method: the role of dilution in the determination of peptic activity. Gastro enterology 5: