INTRADUODENAL HYDROLYSIS OF GELATIN AS A MEASURE OF PROTEIN DIGESTION IN NORMAL SUBJECTS AND IN PATIENTS WITH MALABSORPTION SYNDROMES

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1 GASTROENTEROLOGY Copyright 1968 by The Williams & Wilkins Co. Vol. 54, No.3 Printed in U.S.A. INTRADUODENAL HYDROLYSIS OF GELATIN AS A MEASURE OF PROTEIN DIGESTION IN NORMAL SUBJECTS AND IN PATIENTS WITH MALABSORPTION SYNDROMES JAMES J. CERDA, M.D., FRANK P. BROOKS, M.D., AND DARWIN J. PROCKOP, M.D., PH.D. Department of Medicine, University of Pennsylvania, The Philadelphia General Hospital, ad The Gastrointestinal Research Laboratory, Pennsylvania Hospital, Philadelphia, Pennsylvania It is generally accepted that during digestion proteins are completely hydrolyzed to amino acids before being absorbed from the gastrointestinal tract,2 but several reports 3-8 have suggested that some peptides are absorbed from the gastrointestinal tract under normal conditions. Absorption of peptides has been clearly demonstrated after ingestion of gelatin by normal adult subjects, and up to 8% of the hydroxyproline found in gelatin is absorbed in a peptide-bound form and then excreted in a peptide-bound form in urine. 6, 7 Collagen, which is the native form of gelatin, is the only animal protein which contains significant amounts of the amino acid, hydroxyproline. Essentially all of the endogenous hydroxyproline in animal tissues and fluids is in collagen or in peptides or free hydroxyproline derived from the degradation of collagen. 9 A specific chemical assay is available to measure free and Received July 21, Accepted November 14, Address request for reprints to: Dr. Darwin J. Prockop, Philadelphia General Hospital, 34th Street and Curie A venue, Philadelphia, Pennsylvania This work was supported in part by Grants FR-40, FR-107, FR-5590, and HD-I83 from the National Institutes of Health, United States Public Health Service. A preliminary report on the work has been published,' and a partial report was presented at the Eastern Meeting of the American College of Physicians, December 1, 1966, and at the Gastroenterological Research Forum, Colorado Springs, Colorado, May 25, The authors gratefully acknowledge the expert technical assistance of Miss Nina A. Shopa. 358 peptide-bound hydroxyproline,lo, 11 and therefore it is possible to observe the effect of feeding gelatin on the fasting levels of free and peptide hydroxyproline in plasma, urine, and other body fluids. In the present study a double lumen tube was used to infuse gelatin into the duodenum and to aspirate duodenal contents after the infused solution had traversed 25 cm. The digestion of the gelatin was then studied by measuring the protein-bound, peptidebound, and free hydroxyproline in the duodenal aspirates (fig. 1). Materials and Methods Subjects. Studies were performed on 16 subjects: 5 normal subjects with no history of gastrointestinal disease, 7 patients with pancreatic insufficiency, and 4 patients with adult celiac disease. The diagno'lis of pancreatic insufficiency was established by a documented previous history of acute pancreatitis, stool fat excretion of greater than 5 g per 24 hr., decreased response in duodenal bicarbonate concentration to stimulation with secretin, and a normal small bowel biopsy. All of the patients with pancreatic insufficiency had a significant decrease in stool fat after treatment with pancreatic extracts. The diagnosis of adult celiac disease was established by history of weight loss, steatorrhea, normal secretin test, jejunal biopsy demonstrating total villous atrophy, and a positive therapeutic response to a gluten-free diet. Infusion and aspiration of test solution. Subjects were placed on a gelatin-free diet for 2 days prior to the study. They were fasted overnight and until the infusion of the test solution was completed. A double lumen polyvinyl tube' was used to infuse the gelatin test solution into the duodenum and to aspirate the duodenal contents 25 em below the point of infusion. The double

2 March 1968 HYDROLYSIS OF GELATIN 359 lumen tube was assembled by placing a polyvinyl tube (Portland Plastics, Ltd., Long Island, N. Y.) with an inner diameter of 2.2 mm and an outer diameter of 3.0 mm in a second tube with an inner diameter of 4.0 mm and an outer diameter of 6.0 mm. The distal 25 cm of the space between the two tubes was sealed with Duco cement (DuPont Chemical Company) and 12 openings of about 1 mm diameter were cut in the wall of the outer tube just above the cement seal. The tube was weighted with a Rehfuss tip, and its passage was observed fluoroscopically until the tip was at the ligament of Treitz. The ph of the initial aspirate was measured to verify that the tube was in the duodenum. The test solution consisted of 25 g of gelatin (finely granular form; Fisher Scientific), 9 g of NaCI, and 50 mg of phenol red in 1 liter of water. The ph of the final solution was adjusted to 7. The NaCI was used to keep the solution isotonic, and the phenol red was used as a relatively inert marker to correct for any water flux into or out of the small intestine. The gelatin was dissolved in the solution at 37 C, and the solution was kept in a water bath at 37 C throughout the procedure. The solution was infused continuously for 1 hr through the outer lumen of the tube with a dual infusion and withdrawal pump (Harvard Apparatus Company) at a constant rate of 15.3 ml per min. Constant suction was maintained on the inner lumen with a Wappler-Stedman suprapubic suction pump (American Cystoscopic Company). The aspirate was collected in six 10-min fractions in studies with 13 subjects and in four I5-min fractions in 2 subjects. The volume of aspirate collected in a 10- or I5-min period varied from 15 to 200 ml. The aspirates were collected in ice and frozen until assayed. Assays. Hydroxyproline was assayed by the method of Prockop and U denfriend 10 as modified by Kivirikko et al. ll The assay is specific for free hydroxyproline; protein-bound and peptidebound hydroxyproline do not react. Accordingly, the amount of protein-bound hydroxyproline can be measured as the difference in free hydroxyproline detected before and after hydrolysis of a sample. Samples were hydrolyzed by adding an equal volume of concentrated HCI and autoclaving them in sealed tubes at 110 C and I6-lb pressure for 15 hr. In order to measure hydroxyproline in dialyzable peptides, I-ml aliquots of samples were diluted to 10 ml, and the 10 ml were then placed in cellulose dialyzing tubing. The sample was dialyzed against 40 ml of distilled water in a closed tube with gentle shaking at 4 C for 15 hr. From 1 to 4 ml of the external Infused gelatin (protein-bound hypro) Peptide-bound hypro ----t absorption Free hypro > absorption FIG. 1. Schematic representation of the intraluminal hydrolysis of hydroxyproline (hypro) in gelatin to dialyzable peptides containing hydroxyproline and to free hydroxyproline. solution were used for assay of free hydroxyproline, and about an equa! amount was hydrolyzed for assay of total hydroxyproline. The recoveries of hydroxyproline in the assay procedure were estimated by the addition of internal standards; they ranged from 93 to 101%. The concentration of phenol red in samples was measured by the method of Schedl and Clifton 13 ; the recoveries of phenol red based on addition of internal standards ranged from 95 to 105%. Results Dilution of the infused solution in the duodenal segment. After infusion of the test solution for 10 min, the average concentration of phenol red (fig. 2A) in the aspirate was about 70% of the concentration in the infused solution and, after 20 min of infusion, it was over 80% of. the concentration in the test solution. The maximal values for phenol red concentration, however, were slightly less than the concentration in the infused solution. This observation might be explained by a small dilution of the infused solution but, since assays of hydroxyproline (below) did not indicate any dilution, a more probable explanation is that small amounts of the phenol red were absorbed during passage through the duodenum. 14 Samples of duodenal contents obtained before infusion of the test solution did not contain any significant amounts of hydroxyproline. Also, no significant amounts of hydroxyproline were found in a duodenal aspirate collected for 80 min after administration of secretin, 1.5 mg per kg, to a normal subject. The concentration of total hydroxyproline (fig. 2B) in the aspirate increased at a slightly more rapid rate than the phenol red concentration, and after 20 min it was equal to the concentration of total hydroxyproline in the test solution

3 360 CERDA ET AL. Vol. 54. No.3 Concentration of Phenol Red and Total Hypro in Aspirate (Mean Values a Range) A. o M inules 3500f 3000 g T :t B.. ' ".-'. ;.:.:-, Minules FIG. 2. A. Concentration of phenol red in duodenal aspirates during infusion of the test solution. Symbols indicate mean (0) and range of values in 5 normal subjects and 11 patients with pancreatic insufficiency or adult celiac disease. Shaded area indicates range of values obtained by direct measurements of the test solution. B. Concentration of total hydroxyproline in duodenal aspirates during infusion of the test solution. Symbols indicate mean (0) and range of values in 5 normal subjects and 11 patients with pancreatic insufficiency or adult celiac disease. Shaded area indicates range of values obtained by direct measurements of the test solution. 3000r Peptide Hypro ;-0_'_'_'._0 200J // '" I i // ti i,,.., 1000 c:t", :t' I 0' // \,, 1,' '0 Protein Hypro 500- ;,' ----O Free Hypro O * Minutes FIG. 3. Typical result obtained during infusion of test solution in a normal subject (D. S.). No significant difference in either the phenol red or total hydroxyproline concentration was found between control subj ects and patients with pancreatic insufficiency or adult celiac disease. As discussed below, the results suggest that a constant level of test solution was present in the intestinal segment under examination after about 20 min of infusion, and that there was no significant absorption of free, peptide-bound, or protein-bound hydroxyproline from the infused solution in the duodenal segment. Intraluminal hydrolysis of gelatin in control subjects. Figure 3 presents a typical result obtained when gelatin was infused into normal subj ects. After 10 min of infusion, about half of the hydroxyproline recovered in the aspirate was in a protein. bound form and about half was present as dialyzable peptides containing hydroxyproline. After 20 min of infusion, less than half of the hydroxyproline was recovered in a protein-bound form and, after 20 min of infusion, most of the hydroxyproline in the aspirate was in the form of dialyzable peptides. An equilibrium state was apparently achieved after 30 min of infusion, since there was relatively little change in the level of peptide-bound or protein-bound hydroxyproline after this point. As indi cated in table 1, the concentration of free hydroxyproline was less than 20 p.g per ml in all of the aspirates examined. Aspirates obtained after 1 hi' of infusion in normal subjects contained from 1830 to 2600 p.g per ml of peptide-bound hydroxyproline and from 500 to 970 p.g per ml of nondialyzable protein-bound hydroxyproline.

4 March 1968 HYDROLYSIS OF GELATIN 361 TABLE 1. Intraluminal hydrolysis of protein-bound hydroxyproline in gelatin to free and peptide hydroxyproline Aspirate Total hyproa in Sex/Age I Protein Patient infusion solution hydrolyzed Free Peptide Protein hypro hypro hypro dml Jlg/ml % Control F. B..M < C.M. M < R. B. F < D.8. M < D.8h. M < Pancreatic insufficiency T. G. F < J. D. M < R.W. M < A. J. F < J. B. M < <5 P. T. F < <5 H. K. F < <5 H. K.b F < Adul t celiac disease M.T. F < E.R. F < M. J. F < M. B. F < M. B.c F < a Hypro, hydroxyproline. b Repeat infusion after administration of Viokase. c Repeat infusion 1 year after first examination; small bowel biopsy was still abnormal. On the basis of the hydroxyproline values, 63 to 84% of the infused protein was hydrolyzed to dialyzable peptides in the 5 normal subjects. The mean value was 72% with a standard deviation of ±9.1. Intraluminal hydrolysis of gelatin in patients with pancreatic insufficiency. When the test solution was infused into 7 patients with pancreatic insufficiency, little hydrolysis of the protein-bound hydroxyproline was seen. As in control subjects, the total hydroxyproline in the aspirate increased for 20 to 30 min, and beyond these times the concentration was essentially the same as in the test solution. In the patients with pancreatic insufficiency, however, most of the hydroxyproline in the aspirate was recovered in a nondialyzable form (fig. 4). After 1 hr of infusion, the amount of peptide hydroxyproline varied from 140 to 470 ""g per ml and the protein-bound hydroxyproline varied from 2600 to 2850 ""g per ml (table 1). In 4 patients 10 to 15% of the protein-bound hydroxyproline was converted to dialyzable peptides containing hydroxyproline and in the 3 remaining subjects the conversion was less than 5%. Because the amounts of chromophore obtained in the assay were low, the estimates of the peptide hydroxyproline in patients J. B., P. T., and H. K. represent the maximal amount of peptide hydroxyproline that could be present, and the true values in these patients may have been less than those indicated. Assuming values of 5% for these patients, the mean value for the group was 9% ± 3.9 SD. One of the patients with pancreatic insufficiency (H. K.) was reexamined 2 weeks after the original examination and 1 hr after she received 4 tablets of Viokase (Vio Bin Corporation). Without Viokase therapy, less than 5% of the infused gelatin was hydrolyzed to dialyzable peptides

5 362 CERDA ET AL. Vol. 54, No.3 (fig. 4), but after Viokase 57% of the hydroxyproline in the infused gelatin was hydrolyzed to dialyzable peptides (fig. 5). Intralumtnal hydrolysis of gelatin in patients with adult celiac disease. In 4 patients with adult celiac disease, some hydrolysis of protein-bound hydroxyproline to peptide-bound hydroxyproline was observed, but the degree of hydrolysis was less than in normal controls (table 1). r---o "';,.7." 1;,-;' , " "001 1: '." I ",.2, ,,f i 10001! 500-, O Minutes FIG. 4. Typical result obtained during infusion of test solution in a patient with pancreatic insufficiency (H. K.) , I - I 2000l. il50j I O-... _ Peptide Hypro / Protein Hypro I Minutes FIG. 5. Effect of Viokase therapy on the intraluminal hydrolysis of gelatin in the same patient (H. K.) with pancreatic insufficiency. Control results for this patient are shown in figure r r---, {,J, p.. Protein Hypro, ".. ".0-.._"'--"'-..,,,,,,,,,,,,,, "" ".'..._...-. " \J p' ",e._ Peptide Hypro :./ :t' I /e 1 " e/ 500 I / 1/. Free Hypro O-- Minutes FIG. 6. Typical result obtained during infusion of test solution in a patient (E. R.) with adult celiac disease. One patient was reexamined 1 year after the initial examination, at which time her clinical status had improved as a result of a gluten-free diet, but her small bowel biopsy was still abnormal. As indicated, there was no significant change in the intraluminal hydrolysis of gelatin. A typical result obtained in these patients is shown in figure 6. As with all of the other subj ects examined, the concentrations of peptide and protein-bound hydroxyproline increased for 20 or 30 min, and then remained relatively constant thereafter. The concentration of dialyzable peptide hydroxyproline in the aspirate at 1 hr varied between 1080 and 1500 p.g per ml, and the protein-bound hydroxyproline varied from 1530 p.g per ml to 2020 p.g per ml. The concentration of peptide hydroxyproline was greater than in any of the patients with pancreatic insufficiency, but in contrast to normal subjects the amount of peptide-bound hydroxyproline was always less than the amount of protein-bound hydroxyproline. The calculated percentage of protein hydrolyzed to dialyzable peptides ranged from 35 to 48, and the mean value was 43%. The standard deviation for the five values shown was 5.5%, and the values for the patients with celiac disease differed from the values in the normal subjects with a P value of less than 0.01.

6 March 1968 HYDROLYSIS OF GELATIN 363 Discussion Several difficulties are encountered in an attempt to study the digestion of proteins in the intestine by measuring either the proteolytic enzymes in intestinal secretions or the release of peptides and amino acids. during the intraluminal hydrolysis of protem. Measurements of intraluminal enzyme c.oncentrations are complicated by changes m the volume of the intestinal juices and in intestinal motility which make it difficult to obtain meningful eaurements. The hydrolysis of proteins IS difficult to study within the intestine because of these factors and because the large amounts of amino acids and other substances in intestinal fluids interfere with most assays for exogenous proteins peptides, or amino acids. ' The results indicate that the intraluminal hydrolysis of gelatin can be studied in human subjects by the technique employed here: Tere was essentially no hydroxyprolme m endogenous duodenal fluids and it was possible to assay specific all; the exogenous protein-bound, peptide-bound, and free hydroxyproline which appeared in duodenal aspirates after infusion of gelatin. Also, it was possible to select the distance between openings of the double lumen tube so that significant hydrolysis of gelatin to dialyzable peptides occurred in normal subjects, but there was no net absorption of the hydroxyproline, and there was no significant concentration or dilution of the test solution in either the normal subjects or the patients. The effects of changes in intestinal motility were not examined in detail, but, since the concentrations of phenol red and total hydroxyproline in the aspirates increased at about the same rate over the first 20 to 30 min in all subjects, the results suggest that marked differences in intestinal motility were not encountered. From 63 to 84% of the infused protein was hydrolyzed to dialyzable peptides in normal subjects. It is probable that if a longer segment had been examined, further hydrolysis of protein-bound hydroxyproline to peptide-bound and free hydroxyproline would have been observed and that absorption of significant amounts of the hydroxyproline in the test solution would have occurred. Use of a longer distance between openings of the tube might be advantageous for some purposes but the hydrolysis observed over a 25-c distance appeared to be sufficient to compre values obtained in control subjects with.th.ose obained in patients with pancreatlc msufficlency and celiac disease. A.marked defect in the hydrolysis of glatm was demonstrated in all 7 patients with pancreatic insufficiency. The 7 patients w.ho wee chosen for the study all had the diagnosis of pancreatic insufficiency clearly demonstrated by other criteria but, since the extent of hydrolysis in the patients was less than one-fourth of that observed in ormal s.ubjects, it seems likely that patients with lesser degrees of pancreatic insufficiency could have been detected by t?e test.. The egree of hydrolysis in patients with mlld pancreatic insufficiency would probably be similar to that observed in patients with adult celiac disease, but a normal small bowel biopsy should make it possible to distinguish pancreatic insufficiency from celiac disease. The 4 patients with adult celiac disease demon.strated a partial defect in the hydrolyi of gelatm to dialyzable peptides contammg hydroxyproline. The results did not establish a specific explanation for this observation, but several hypotheses can be offered. For example, the partial defect in the hydrolysis of protein in these patients may be a direct result of the mucosal changes which occur in celiac disease. Recent reports have suggested that disaccharides are hydrolyzed on the cell surface of the intestine by mucosa-bound enzymes, and the amount of leucine amino peptidase in the brush border of intestinal villi has been shown to be decreased in patients with idiopathic steatorrheap. 18 Aternate explanations are that the patients with adult celiac disease may have some secondary pancreatic insufficiency19 or that because of the mucosal changes in the duodenum, there is a decreased release of secretin during infusion of gelatin. We are currently exploring the latter possibility

7 364 CERDA ET AL. Vol. 54, No.3 by measuring the intraluminal hydrolysis of gelatin in patients with celiac disease before and after administration of secretin. In earlier studies, Thiel, Benoit, and Watten 20 administered a test solution of gelatin and xylose orally, and they found that the amount of hydroxyproline excreted in the urine was decreased in 4 patients with malabsorption syndromes. Bronstein, Haeffner, and Kowlessar21 studied both serum and urine levels of free and peptide hydroxyproline after ingestion of gelatin, and they found that the increases of peptide hydroxyproline in plasma and urine were delayed in 2 of 3 patients with pancreatic insufficiency and in 2 of 3 patients with adult celiac disease. The test procedure employed here offers a more direct method for evaluating protein digestion in patients, and the values obtained in patients were consistently different from those observed in normal controls. The procedure is probably too cumbersome to be employed in the routine evaluation of malabsorption syndromes, but it offers a precise method for the investigation of protein digestion in these conditions. Summary Gelatin was used as a test solution to study the intraduodenal hydrolysis of protein in normal subjects and in patients with malabsorption syndromes. A double lumen tube was used to infuse gelatin into the duodenum and to aspirate duodenal contents after the infused solution had traversed 25 cm. The hydrolysis of gelatin was then studied by measuring the conversion of protein-bound hydroxyproline to peptide-bound and free hydroxyproline in the duodenal aspirates. In 5 normal subjects, 63 to 84% of the hydroxyproline in the infused gelatin was digested to dialyzable peptides after flowing through the measured segment of the duodenum. In 7 patients with pancreatic insufficiency, less than 15% of the infused gelatin was digested to dialyzable peptides, demonstrating a marked defect in the hydrolysis of gelatin. In 4 patients with adult celiac disease, 35 to 48% of the infused gelatin was digested to dialyzable peptides, indicating a partial defect in hydrolysis of gelatin. The procedure is probably too cumbersome to be employed in the routine evaluation of malabsorption syndromes, but it offers a precise method for the investigation of protein digestion in these conditions. REFERENCES 1. Cerda, J. J., F. P. Brooks, 1. S. Weisner, and D. J. Prockop Digestion and absorption of hydroxyproline pep tides in malabsorption syndromes. Clin. Res. 14: Matthews, D. M., and L. Laster Absorption of protein digestion products. A review. Gut 6: Wiggens, D. S., and J. M. Johnston The absorption of peptides. Biochim. Biophys. Acta 32: Newey, H., and D. H. Smyth Intracellular hydrolysis of dipeptides during intestinal absorption. J. Physiol. (London) 152: Ziff, M., A. Kibrick, E. Dresner, and H. J. Gribetz Excretion of hydroxyproline in patients with rheumatic and non-rheumatic diseases. J. Clin. Invest. 35: Prockop, D. J., and A. Sjoerdsma Significance of urinary hydroxyproline in man. J. Clin. Invest. 40: Prockop, D. J., H. Keiser, and A. Sjoerdsma Gastrointestinal absorption and renal excretion of hydroxyproline pep tides. Lancet 2: Pisano, J. J., J. Freedman, and L. Cohen Increased urinary excretion of fjaspartylhistidine in response to certain dietary proteins. Fed. Proc. 22: Prockop, D. J., and K. 1. Kivirikko Relationship of hydroxyproline excretion to collagen metabolism. Ann. Intern. Med. 66: Prockop, D. J., and S. Udenfriend A specific method for analysis of hydroxyproline in tissues and urine. Anal. Bioehem. 1: Kivirikko, K. 1., O. Laitinen, and D. J. Prockop Modifications of a specific assay for hydroxyproline in urine. Anal. Biochem.19: Fordtran, J. S., P. H. Clodi, K. H. Soergel, and F. J. Inglefinger Sugar absorption tests, with special reference to 3-0-methyld-glucose and d-xylose. Ann. Intern. Med. 57:

8 March 1968 HYDROLYSIS OF GELATIN Schedl, H. P., and J. A. Clifton Small intestinal absorption of steroids. Gastroenterology 41: Schedl, H. P Use of polyethylene glycol and phenol red as unabsorbed indicators for intestinal absorption studies III man. Gut '1: Gray, G. M., and F. J. Inglefinger Intestinal absorption of sucrose in man: the site of hydrolysis and absorption. J. Clin. Invest. 44: Gray, G. M., and F. J. Inglefinger Intestinal absorption of sucrose in man: interrelation of hydrolysis and monosaccharide product absorption. J. Clin. Invest. 45: Spiro, H. M., M. I. Filipe, J. S. Stewart, C. C. Booth, and A. G. E. Pearse Functional histochemistry of the small bowel mucosa in malabsorptive syndromes. Gut 5: Riecken, E. 0., J. S. Stewart, C. C. Booth, and A. G. E. Pearse A histochemical study on the role of lysosomal enzymes in idiopathic steatorrhea before and during a gluten-free diet. Gut 1: Dreiling, D. A The pancreatic secretion in the malabsorption syndrome and related malabsorption states, p. 69. In A. Adleraberg [ed.], Symposium: the malabsorption syndrome. Grune and Stratton, New York. 20. Thiel, G. B., F. L. Benoit, and R. H. Watten An oral gelatin-xylose test for estimating pancreatic proteolytic activity. Amer. J. Dig. Dis. 8: Bronstein, H. D., L. J. Haeffner, and O. D. Kowlessar The significance of gelatin tolerance in malabsorptive states. Gastroenterology 50: