Gut: first published as 10.1136/gut.21.8.662 on 1 August 1980. Downloaded from Gut, 1980, 21, 662-668 Immunoelectrophoretic studies on human small intestinal brush border proteins: cellular alterations in the levels of brush border enzymes after jejunoileal bypass operation H SKOVBJERG, E GUDMAND-H0YER, 0 NOREN, AND H SJOSTROM From the Department ofBiochemistry C, The Panum Institute, Medical-gastroenterological Department C, and Surgical-gastroenterological Department D, Herlev Hospital, University of Copenhagen, Denmark SUMMARY The amounts of lactase (EC 3.2.1.23), sucrase (EC 3.2.1.48), maltase (EC 3.2.1.20), microvillus aminopeptidase (microsomal EC 3.4.11.2), and dipeptidyl peptidase IV (EC 3.4.14. x) in biopsies from proximal jejunum and distal ileum were studied by quantitative crossed immuno- electrophoresis and enzymatic assays in obese patients one and six months after jejunoileal bypass operation and compared with peroperative levels. They were related to DNA and protein content. The protein/DNA ratio fell 28-43 % postoperatively. Except for ileal lactase and sucrase all enzymes showed decreased levels when expressed per mg protein and an even more pronounced decrease when related to DNA. Lactase and sucrase levels in ileum were increased or unchanged. A constant correlation between the amount of immunoreactive enzyme protein and enzymatic activity was shown for all enzymes except maltase. The results suggest that the bypass operation is followed http://gut.bmj.com/ by an increased amount of enterocytes devoid of or low in enzymatic activity and protein content. The amounts of lactase and sucrase in ileum are increased in relation to the other enzymes. No immunoreactive enzymes with zero or depressed activity were detected. Jejunoileal bypass operation frequently used in the to an altered amount of active enzyme molecules in on September 29, 2021 by guest. Protected copyright. treatment of morbid obesity results in malabsorption the cells, or to the presence of more or less active and weight loss. The increase in length and mucosal enzyme molecules. Experimental studies on bypass surface of the functioning shunt seems to be im- operated rats7 have shown an increase in the DNA portant for the weight stabilisation that usually content per segment small intestine and a falling occurs 12 to 18 months postoperatively. Increase of activity of brush border enzymes expressed per mg villus height and epithelial cell hyperplasia has been DNA indicating an increased amount of cells described after the operation.'2 Changes in the devoid of or low in enzymatic activity. These cells specific activity of some of the brush border enzymes appear either as crypt cells or immature cells on the (disaccharidases and peptidases) which play an villi. Corresponding studies on human intestine important role in the final digestion have been have not been undertaken. described,' 3 4-6 but the results are conflicting, The present study investigates the cellular changes probably because not only the cellular enzyme in the small intestinal brush border enzymes by the content but also the mucosal protein change post- use of quantitative immunoelectrophoresis and operatively. DNA measurement in combination with measure- The reported changes in brush border enzyme ments of enzymatic activity and protein. Quantita- activity can be caused by an alteration in the relative tive immunoelectrophoresis, which earlier has been number of enterocytes or by a change in enzymatic shown to be a valuable method in the study of activity at a cellular level. Such changes might be due human brush border enzymes,8 gives an expression of the amount of enzyme protein independent of the Received for publication 6 February 1980 enzymatic activity. This enables an estimation of 662 Gut: first published as 10.1136/gut.21.8.662 on 1 August 1980. Downloaded from Immunoelectrophoretic studies on human small intestinal brush border proteins 663 possible enzyme molecules with altered enzymatic patients with 12.5 cm jejunum and 375 cm ileum activity. Moreover, changes in the internal relations (type IL) left in continuity. Peroperative biopsies between the enzymes measured can be more precisely from the points of division-that is, proximal determined, as they are quantified on the same im- jejunum and distal ileum-were obtained in all munoelectrophorectic plate. The method also, for patients. One to two and six to eight months post- the first time, allows changes occurring in the operatively peroral biopsies were taken 25 cm below amount of brush border maltase to be estimated, as the ligament of Treitz-that is, jejunal biopsies the enzymatic estimation of maltase activity also from patients with type I and ileal biopsies from includes the activity of at least the sucrase-isomaltase patients with type II operation. Nine patients with complex. type I and seven with type 1I operation had biopsies taken one to two months postoperatively, while Methods nine patients with type l and six with type IL operation had biopsies taken six to eight months postopera- PATIENTS tively. All biopsies were frozen in dry ice immedi- Twenty patients having jejunoileal shunt operation ately after removal. performed as a part of The Danish Obesity Project were investigated. Eleven patients were operated TECHNIQUE according to Payne and De Wind9 with 37.5 cm From the peroperative biopsies mucosal samples jejunum and 12.5 cm ileum (type I) and nine (3-16 mg) were obtained by careful scraping. The Jejunum Ileum Jejunum Ileum Lactase l 1IAt\** ** NS http://gut.bmj.com/ z c Fig. 1 Changes in amount oj E enzyme protein for each patient cn ,estimated in crossed E immunoelectrophoresis as area U Sucrase Microvillus Aminopeptidase under the precipitate per mg DNA .at operation, one to two and six to on September 29, 2021 by guest. Protected copyright. eight months after jejunoileal shunt operation. Jejunal values are from patients with type I operation, ileal values from patients with type II operation. NS: not significant. The P values are obtained by testing the z postoperative values against the 0 peroperative values. Interrupted lines: in these patients the one to E CN~ two months postoperative biopsies E Dipeptidyl Peptidase I1: were not taken. U OP 1 6 OP 1 6NS *** = p< 0.01 200 * * = P< 0.02 *= P< 0.05 100 0 OP 1 6 OP 6 Gut: first published as 10.1136/gut.21.8.662 on 1 August 1980. Downloaded from 664 Skovbjerg, Gudmand-Hoyer, Noren, and Sjostrirm Maltase Lactase Sucrase Jejunum Ileum Jejunum Ileum Jejunum Ileum NS * *..NS ..NS .***NS NS c 0 4 E Fig. 2 Changes in amount of enzyme' protein for each patient estimated in 2 [ 2 l 6 crossed immunoelectrophoresis as area under the precipitate per mg protein at operation, one to two and I: un six to eight months after jejunoileal OP l 6 Of '1 6 OP 16 OP 16 OP 16 OP 16 shunt operation. Jejunal values are from patients with type I operation, Microvillus Aminopeptidase Dipeptidyl Peptidase XI ileal values from patients with type 1! Jejunum Ileum Jejunum Ileum operation. The bars represent the OP l 6OPNS NS*6PNSNS6Ol median values. NS: not significant. c 8 The P values are obtained by testing the postoperative values against the 0 *** =P 0.01 a. peroperative values. w. cm =p<0.02 E 4 * =p< 0.05 E 2- U ck:WL OP 16 OP 16 0P016 OP 16 mucosal material was then handled as the peroral standard. DNA concentration was measured using http://gut.bmj.com/ biopsies. Each biopsy was homogenised and the the fluorometric method of Kissane and Robins.13 brush border proteins solubilised and quantified in Calf thymus DNA was used as a standard. Extrac- crossed immunoelectrophoresis as earlier described.8 tion of lipids was omitted, as it resulted in irrepro- To summarise briefly: each sample was homogenised ducible loss of material and as the contribution of in 40 ,ul of a 2% Triton X-100 solution and incu- fluorescence from lipids in the homogenate was bated for one hour at 4°C. Papain at a final concen- shown to be less than 8% of the measured value. tration of 2.5 mg/ml was added and 15 pl removed The statistical evaluations were performed with the for enzymatic analysis. The rest of the homogenate Mann-Whitney U test and the Wilcoxon test for on September 29, 2021 by guest. Protected copyright. was incubated for 15 minutes at 37°C and subse- paired data. quently centrifuged at 50.000X g for two hours. The supernatant was analysed in crossed immuno- Results electrophoresis. The precipitates, earlier identified by enzymatic staining,10 were stained by Coomassie As shown earlier8 the solubilisation procedure re- Brillant Blue 250 and their areas expressed in cm2 by leases the studied enzymes almost completely from use of an electronic integrator. the brush border membranes. The mean rest activity Lactase (EC 3.2.1.23), sucrase (EC 3.2.1.48), and of microvillus aminopeptidase in the pellet after maltase activities were assayed in the homogenate solubilisation was for each patient group 2% (range- using their corresponding disaccharides as sub- 0-5%) of the total activity. The protein/DNA ratio strates.1" The activities of microvillus amino- in the peroperative biopsies obtained by mucosal peptidase (microsomal, EC 3.4.11.2) and dipeptidyl scrapings was found to be of the same magnitude peptidase IV (EC 3.4.14. X) were determined using as in peroral biopsies from the ligament of Treitz in L-alanine-p-nitroanilide and glycyl-L-proline-p- patients without gastrointestinal disease. This nitroanilide as substrate respectively.10 As a control indicates that the mucosal scrapings are comparable of the solubilisation procedure the microvillus with the peroral biopsies where their content of aminopeptidase activity was determined in the villi and crypt cells is concerned.