Studies on the Mechanism of Destruction in Coeliac Disease By
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Gut: first published as 10.1136/gut.5.4.295 on 1 August 1964. Downloaded from Gut, 1964, 5, 295 Studies on the mechanism of destruction of the toxic action of wheat gluten in coeliac disease by crude papain M. MESSER, CHARLOTTE M. ANDERSON, AND LOIS HUBBARD From the Gastroenterological Research Unit, Royal Children's Hospital Research Foundation, Melbourne, Australia EDITORIAL SYNOPSIS The toxic action of wheat gluten on two patients was eliminated after pre- digestion of gluten by crude papain. This change is thought to be due to an enzyme which liberates ammonia from gluten. It is not thought that this particular mechanism operates in the normal intestinal cell but it is envisaged that one of the peptide bonds of the coeliac active constituent (possibly a N-glutaminyl peptide) is normally split by a specific intestinal peptidase which is defective in coeliac patients. Previous authors have shown that the harmful latex (papain and chymopapain) was fed to three action of dietary wheat gluten in coeliac disease or coeliac patients. non-tropical sprue is not eliminated by predigesting We have also carried out certain investigations the gluten with either pancreatin or pepsin or into the chemical mechanism of the digestion of trypsin (Alvey, Anderson, and Freeman, 1957; gluten by crude papain, concerned mainly with the http://gut.bmj.com/ Frazer, Fletcher, Ross, Shaw, Sammons, and question of the origin of the free ammonia which is Schneider, 1959; Krainick, Mohn, and Fischer, 1959; formed during this digestion (Krainick et al., 1959). van Roon, Haex, Seeder, and de Jong, 1960). Of These investigations led to the discovery of a new various enzymes from animal sources, only an enzyme contained in papaya latex which acts extract of hog intestinal mucosa has so far been specifically on N-glutaminyl peptides (Messer, 1963); shown to abolish the toxic action of gluten in the possible role of this enzyme in the detoxification coeliac disease (Frazer, 1956). ofgluten by crude papain was given special attention. on October 2, 2021 by guest. Protected copyright. It has been reported, however (Krainick et al., 1959), that after wheat gluten has been digested by PATIENTS, METHODS, AND MATERIALS crude papain (a commercial product prepared from the latex of the unripe papaya) it is no longer COELIAC PATIENTS The methods used in the diagnosis of harmful when fed to coeliac patients. This pheno- coeliac patients, the collection of their stools during not feeding investigations, and the estimation of stool fat menon has been investigated further, and it is were the same as those described in pIevious papers not known whether the 'detoxification' is caused by (Anderson, Frazer, French, Gerrard, Sammons, and papain itself or by another enzyme contained in the Smellie, 1952; Messer and Anderson, 1961). Enzymatic crude product, such as chymopapain (Jansen and gluten digests were administered in three doses per day, Balls, 1941) or an enzyme acting on peptides. either neat or mixed with milk. When the patients were The present studies were undertaken in order to on a gluten-containing diet, meals were prepared by the confirm and extend these results of Krainick et al. diet kitchen to contain about 9 g. wheat gluten per day. (1959), in the hope that knowledge of the mechanism of action of crude papain on gluten might yield GLUTEN Gliadin-enriched wheat gluten was supplied by Barret's Food Co. Pty. Ltd., Melbourne, who state that information concerning the chemical structure of it was prepared by stirring commercial dried gluten in the coeliac-active constituent of the protein. 0-01 M acetic acid, centrifuging, and precipitating the To this end we have conducted experiments in gluten from the supernatant solution with sodium which gluten which had been predigested either by chloride, followed by freeze-drying. Its amide content was crude papain or by purified constituents of papaya 2-76 mmoles per gram dry weight. 295 296 M. Messer, Charlotte M. Anderson, and Lois Hubbard Gut: first published as 10.1136/gut.5.4.295 on 1 August 1964. Downloaded from PEPTIDES L-Glutaminyl-L-asparagine was supplied by digest was then filtered and the clear, orange filtrate Dr. J. M. Swan, C.S.I.R.O., Melbourne, and L-glutaminyl adjusted to pH 10 and evaporated in vacuo at 37°C. to a L-leucine by Drs. G. Amiard and R. Heymes, Roussel- volume of 1 1. by means of a rotary evaporator. The dark Uclaf, Paris. Small amounts of the corresponding orange ammonia- and toluene-free product was adjusted pyrrolidone carboxylyl peptides were prepared for use as to pH 6 5, filtered and stored at - 20C. until use. chromatographic markers by heating the N-glutaminyl The total amount of insoluble material (mainly lipid) peptides for 60 minutes at 100°C. in 0-1 M phosphate removed during the procedure was about 5 g.; the final buffer, pH 8 (Waelsch, 1952). Glycyl-L-glutamine was product was therefore about 9.5% with respect to digested supplied by Dr. J. M. Swan. All other peptides were gluten. obtained from Mann Research Laboratories, New York. CRYSTALLINE PAPAIN DIGEST This was prepared in the ENZYMES Crude papain was obtained from E. Merck same way, except that 2 g. crystalline papain was used A.G., Darmstadt, and crude ficin and bromelin from per 100 g. gluten. The final product was yellow. Mann Research Laboratories; crystalline papain, pepsin, trypsin, and chymotrypsin were products of Sigma CHYMOPAPAIN DIGEST Since native gluten was found to Chemical Co., St. Louis. be digested by chymopapain with great difficulty, it was Semi-pure, non-crystalline chymopapain was prepared predigested by pepsin; 1 g. crystalline pepsin was dissolved by the following modification of the method of Jansen in 2 1. cysteine (5 mM) and E.D.T.A. (1 mM), pH 2-0, and Balls (1941). Finely ground granular dried papaya and 100 g. gluten was added. After two hours' stirring latex (Wheeler and Huisking Pty. Ltd., London), 80 g., the mixture had become homogeneous and was adjusted was stirred with 800 ml. water for 30 minutes. The to pH 5-5. Cysteine (5 mM), and E.D.T.A. (1 mM), suspension was centrifuged and the supernatant solution pH 5.5, 21., containing 10 g. dissolved chymopapain, was adjusted to pH 2-0 and held at 37°C. for 80 minutes. It added and the mixture readjusted to pH 5.5. The rest of was then centrifuged and the supernatant solution the procedure was as described for crude papain. The adjusted to pH 40 and half saturated with sodium final product was orange. chloride. After centrifugation the clear supernatant Measurements of the formation of ninhydrin-reactive solution was fully saturated with sodium chloride and material showed that under the above conditions the adjusted to pH 2-0. It was stood in the refrigerator digestion was over 90% complete in each case. overnight and then centrifuged. The white precipitate was chymopapain (about 10 g.). The main differences OTHER METHODS The amide content of gluten and of between this method and that of Jansen and Balls (1941) gluten digests was estimated by alkaline hydrolysis at are the use of dried instead of fresh papaya latex as the room temperature (Stegemann, 1958). starting product and the 80-minute treatment at pH 2-0 at Enzyme protein was determined by the biuret method 370C. This treatment was necessary in order to destroy all described by Aldridge (1957). http://gut.bmj.com/ deamidase and glutamine cyclotransferase activities (see Free ammonia and ninhydrin-reactive material in the Discussion). The treatment also inactivated papain. The gluten digests were estimated as follows. Of the digest, chief contaminant of our chymopapain is probably 0-10 ml. was treated with 0-1 ml. saturated K2C03 lysozyme (Ebata and Yasunobu, 1962). solution in the outer well of a Conway microdiffusion The activities of the crude papain, chymopapain, and unit and the ammonia liberated into 1-00 ml. 0.1 N crystalline papain towards benzoyl arginine amide were sulphuric acid. After 60 minutes the acid was analysed measured at 25°C., pH 7-2 in the presence of 5 mM for ammonia (Brown, Duda, Korkes, and Handler, 1957) cysteine, 1 mM ethylene diamine tetra-acetic acid and the contents of the outer well diluted to 25-0 ml. and on October 2, 2021 by guest. Protected copyright. (E.D.T.A.), and 50 mM substrate. They were found to be analysed for ninhydrin-reactive material by the method 0 40, 0.33, and 2-7 jgmoles/min./mg. enzyme protein, of Cocking and Yemm (1954). Standard mixtures of respectively. ammonium chloride and leucine were run through the whole procedure. PREPARATION OF ENZYMATIC DIGESTS OF GLUTEN Paper chromatography (descending) was carried out on Whatman no. 1 paper with the following solvent CRUDE PAPAIN DIGEST Crude papain, 40 g., was stirred systems; n-butanol: acetic acid : water (4:1:1), s-butanol: for 30 minutes in 2 1. cysteine (10 mM) and E.D.T.A. t-butanol: 2-butanone: water (4:4:8:5) and propanol: (2 mM), pH 7*5. The suspension was filtered and the water (4:1). For two-dimensional chromatography the filtrate, which contained about 20 g. dissolved crude first two systems were used (Ambe and Tappel, 1961). papain, was vigorously stirred by a magnetic bar while 100 g. gluten was slowly sprinkled onto the surface; this RESULTS OF FEEDING INVESTIGATIONS procedure prevented the formation of lumps. During the WITH COELIAC PATIENTS addition of gluten the pH was maintained above 5.5 by dropwise additions of 5 M NaOH solution. Thirty PATIENT 1 This boy had been diagnosed as suffering minutes after all the gluten had been added the mixture from coeliac disease at the age of 13 months.