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Gut, 1980, 21, 662-668
Immunoelectrophoretic studies on human smallintestinal brush border proteins: cellular alterationsin the levels of brush border enzymes afterjejunoileal bypass operationH SKOVBJERG, E GUDMAND-H0YER, 0 NOREN, AND H SJOSTROM
From the Department of Biochemistry 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 bypassoperation 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 enzymesshowed decreased levels when expressed per mg protein and an even more pronounced decreasewhen related to DNA. Lactase and sucrase levels in ileum were increased or unchanged. A constantcorrelation between the amount of immunoreactive enzyme protein and enzymatic activity wasshown for all enzymes except maltase. The results suggest that the bypass operation is followedby 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. Noimmunoreactive enzymes with zero or depressed activity were detected.
Jejunoileal bypass operation frequently used in thetreatment of morbid obesity results in malabsorptionand weight loss. The increase in length and mucosalsurface of the functioning shunt seems to be im-portant for the weight stabilisation that usuallyoccurs 12 to 18 months postoperatively. Increase ofvillus height and epithelial cell hyperplasia has beendescribed after the operation.'2 Changes in thespecific activity of some of the brush border enzymes(disaccharidases and peptidases) which play animportant role in the final digestion have beendescribed,' 3 4-6 but the results are conflicting,probably because not only the cellular enzymecontent but also the mucosal protein change post-operatively.The reported changes in brush border enzyme
activity can be caused by an alteration in the relativenumber of enterocytes or by a change in enzymaticactivity at a cellular level. Such changes might be due
Received for publication 6 February 1980
to an altered amount of active enzyme molecules inthe cells, or to the presence of more or less activeenzyme molecules. Experimental studies on bypassoperated rats7 have shown an increase in the DNAcontent per segment small intestine and a fallingactivity of brush border enzymes expressed per mgDNA indicating an increased amount of cellsdevoid of or low in enzymatic activity. These cellsappear either as crypt cells or immature cells on thevilli. Corresponding studies on human intestinehave not been undertaken.The present study investigates the cellular changes
in the small intestinal brush border enzymes by theuse of quantitative immunoelectrophoresis andDNA measurement in combination with measure-ments of enzymatic activity and protein. Quantita-tive immunoelectrophoresis, which earlier has beenshown to be a valuable method in the study ofhuman brush border enzymes,8 gives an expressionof the amount of enzyme protein independent of theenzymatic activity. This enables an estimation of
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Immunoelectrophoretic studies on human small intestinal brush border proteins
possible enzyme molecules with altered enzymaticactivity. Moreover, changes in the internal relationsbetween the enzymes measured can be more preciselydetermined, as they are quantified on the same im-munoelectrophorectic plate. The method also, forthe first time, allows changes occurring in theamount of brush border maltase to be estimated, asthe enzymatic estimation of maltase activity alsoincludes the activity of at least the sucrase-isomaltasecomplex.
Methods
PATIENTSTwenty patients having jejunoileal shunt operationperformed as a part of The Danish Obesity Projectwere investigated. Eleven patients were operatedaccording to Payne and De Wind9 with 37.5 cmjejunum and 12.5 cm ileum (type I) and nine
patients with 12.5 cm jejunum and 375 cm ileum(type IL) left in continuity. Peroperative biopsiesfrom the points of division-that is, proximaljejunum and distal ileum-were obtained in allpatients. One to two and six to eight months post-operatively peroral biopsies were taken 25 cm belowthe ligament of Treitz-that is, jejunal biopsiesfrom patients with type I and ileal biopsies frompatients with type II operation. Nine patients withtype I and seven with type 1I operation had biopsiestaken one to two months postoperatively, whilenine patients with type l and six with type IL operationhad biopsies taken six to eight months postopera-tively. All biopsies were frozen in dry ice immedi-ately after removal.
TECHNIQUEFrom the peroperative biopsies mucosal samples(3-16 mg) were obtained by careful scraping. The
Jejunum Ileum Jejunum Ileum
Fig. 1 Changes in amount ojenzyme protein for each patient,estimated in crossedimmunoelectrophoresis as areaunder the precipitate per mg DNA.at operation, one to two and six toeight months after jejunoileal shuntoperation. Jejunal values are frompatients with type I operation, ilealvalues from patients with type IIoperation. NS: not significant. The Pvalues are obtained by testing thepostoperative values against theperoperative values. Interruptedlines: in these patients the one totwo months postoperative biopsieswere not taken.
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Skovbjerg, Gudmand-Hoyer, Noren, and Sjostrirm
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mucosal material was then handled as the peroralbiopsies. Each biopsy was homogenised and thebrush border proteins solubilised and quantified incrossed immunoelectrophoresis as earlier described.8To summarise briefly: each sample was homogenisedin 40 ,ul of a 2% Triton X-100 solution and incu-bated for one hour at 4°C. Papain at a final concen-
tration of 2.5 mg/ml was added and 15 pl removedfor enzymatic analysis. The rest of the homogenatewas incubated for 15 minutes at 37°C and subse-quently centrifuged at 50.000X g for two hours. Thesupernatant was analysed in crossed immuno-electrophoresis. The precipitates, earlier identifiedby enzymatic staining,10 were stained by CoomassieBrillant Blue 250 and their areas expressed in cm2 byuse of an electronic integrator.
Lactase (EC 3.2.1.23), sucrase (EC 3.2.1.48), andmaltase activities were assayed in the homogenateusing their corresponding disaccharides as sub-strates.1" The activities of microvillus amino-peptidase (microsomal, EC 3.4.11.2) and dipeptidylpeptidase IV (EC 3.4.14. X) were determined usingL-alanine-p-nitroanilide and glycyl-L-proline-p-nitroanilide as substrate respectively.10 As a controlof the solubilisation procedure the microvillusaminopeptidase activity was determined in thepellet. The protein concentration of the homogenatewas measured12 in a small sample taken before ad-dition of papain using bovine serum albumin as a
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Fig. 2 Changes in amount of enzyme'protein for each patient estimated incrossed immunoelectrophoresis asarea under the precipitate per mgprotein at operation, one to two andsix to eight months after jejunoilealshunt operation. Jejunal values arefrom patients with type I operation,ileal values from patients with type 1!operation. The bars represent themedian values. NS: not significant.The P values are obtained by testingthe postoperative values against theperoperative values.
standard. DNA concentration was measured usingthe fluorometric method of Kissane and Robins.13Calf thymus DNA was used as a standard. Extrac-tion of lipids was omitted, as it resulted in irrepro-ducible loss of material and as the contribution offluorescence from lipids in the homogenate wasshown to be less than 8% of the measured value.The statistical evaluations were performed with theMann-Whitney U test and the Wilcoxon test forpaired data.
Results
As shown earlier8 the solubilisation procedure re-
leases the studied enzymes almost completely fromthe brush border membranes. The mean rest activityof microvillus aminopeptidase in the pellet aftersolubilisation was for each patient group 2% (range-0-5%) of the total activity. The protein/DNA ratioin the peroperative biopsies obtained by mucosal
scrapings was found to be of the same magnitudeas in peroral biopsies from the ligament of Treitz inpatients without gastrointestinal disease. Thisindicates that the mucosal scrapings are comparablewith the peroral biopsies where their content ofvilli and crypt cells is concerned.
Peroperative sucrase and lactase levels as measuredby immunoelectrophoresis and enzymatic activitywere significantly greater (P<0-05) in jejunal than
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Immunoelectrophoretic studies on human small intestinal brush border proteins
in ileal biopsies. The levels of maltase, microvillusaminopeptidase, and dipeptidyl peptidase IV seemedlower in proximal jejunum than in distal ileum butthe differences were not statistically significant.The variations in enzyme protein per mg DNA
for each patient for maltase, sucrase, lactase, micro-villus aminopeptidase, and dipeptidyl peptidase IVone to two and six to eight months after operation,compared with the peroperative levels, are seen inFig. 1. In jejunum all the measured enzymes de-creased in parallel during the first postoperativemonths and remained significantly lower than per-operatively six to eight months after the operation.In ileum the levels of lactase and sucrase were notsignificantly changed during the study, while the
other enzymes were decreased in parallel during thefirst postoperative months and remained decreased,though not significantly, six to eight months post-operatively.When expressed as the area under the precipitate
per mg protein (Fig. 2) all enzymes in jejunumtended to decrease. In ileum, lactase and sucraseincreased while the other enzymes decreased almostin parallel.For all the enzymes studied, except maltase, there
was a close correlation between enzymatic activityand amount of enzyme protein irrespective of de-creasing or increasing postoperative activity (Fig. 3).
Figure 4 exemplifies the results by showing theelectrophoretic patterns in jejunal biopsies from the
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Skovbjerg, Gudmand-Hoyer, Noren, and Sjostrom* :; ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.- ......:. ...... :. ;... :iFig. 4 Crossed immunoelectrophoresis of biopsies taken from the proximal part ofjejunum (a) during operation,(b) one month after operation from patient A. The IgG concentration in the gel was 30 [lg/crn2. The amount ofantigen is equivalent to 4 and 15 t£g DNA respectively. L: lactase-phlorizin hydrolase. S: sucrase-isomaltase.M: maltase. A: microvillus aminopeptidase. G: dipeptidyl peptidase IV. AP: alkaline phosphatase (this precipitateis visible only after enzymatic staining).
same patient (type I operation) taken at operationand one month postoperatively (marked in Fig. 1 byA). This patient had a marked fall in lactase (99%),
Table Mean values for ratio mg protein/mg DNA atoperation, one to two months and six to eight monthspostoperatively for jejunum and ileum*
Operation Postoperatively (months)
1-2 6-8
45-8 26 1 29.2Jejunum (19-9-94-3) (16-6-37.3) (13-5-49 3)
n= 18 n=9 n=9P<0-01 P<002
47.8 27-2 34-5Ileum (32-3-71-3) (22-9-34.1) (20.7-51.0)
n= 17 n=7 n=6P<0t)0 NS
*Numbers in parentheses are ranges. The P values apply to differencesbetween operation and one to two or six to eight months postopera-ii ely. NS: not significant.
while the other enzymes were all reduced by ap-proximately 75% one month postoperatively com-pared with the operative level.The Table shows the relation between protein and
DNA content in the biopsies. The 28-43% fall inthe ratio postoperatively is of the same magnitudeas found in the small intestine from bypass operatedrats.7
Discussion
In the present study changes in the amount ofdifferent brush border enzymes have been examinedin relation to changes in DNA and protein content.The 28-43%0 fall in the protein/DNA ratio in-
dicates that the protein content in the averageenterocyte is diminished postoperatively. An in-creased number of dividing crypt cells increasingthe average DNA content in the cells might cont-
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Immunoelectrophoretic studies on human small intestinal brush border proteins 667
tribute to the decreased ratio but could not accountfor all of it. The reduced amount of protein in theenterocytes is in accordance with the findings ofDan0 et al.14 and is probably due to the postoperativeprotein malabsorptive state.1516The almost parallel decrease of all enzymes in
jejunum per mg DNA indicates a decreased amountof enzyme per cell. Morphological studies in rat andhuman1 317 have shown an increased amount ofenterocytes on the villi after bypass operation, whileno alteration in the proportion of enterocytes to thetotal cell number has been described. The fallingenzyme activity per cell indicates the existence of asignificant number of enterocytes devoid of or lowin enzyme content. Whether the patients in additionto an increased migration rate of the enterocytes17and increased cell proliferation'8 have an increasedturnover rate of the enterocytes cannot be settledin this study.
In ileum the levels of lactase and sucrase were un-changed during the period that was studied, whilethe levels of maltase, microvillus aminopeptidase,and dipeptidyl peptidase IV were decreased almostin parallel when expressed per mg DNA. Thisindicates that lactase and sucrase at the cellular levelare increased in relation to the other enzymes, and itsuggests that these disaccharidases are regulatedindependently of the other enzymes. An explanationmight be that there is a relatively increased synthesisof lactase and sucrase due to enhanced intraluminalstimulation by lactose and sucrose, because theshort functioning jejunum (12.5 cm) in these patientsdoes not have enough capacity to split these disac-charides. This result is supported by the concept' 17
that the intraluminal nutrition exerts a major in-fluence on the splitting and absorptive functions ofthe human small intestine. Alternatively, thecatabolism of maltase, microvillus aminopeptidase,and dipeptidyl peptidase IV in ileum might berelatively increased postoperatively. Duodenal juiceincreases the turnover rate of some brush borderenzymes.'9 20 Theoretically, maltase, microvillusaminopeptidase, and dipeptidyl peptidase IV-which, in contrast with lactase and sucrase, seem tohave a higher peroperative level in the trypsin free"distal ileum than in the proximal jejunum-might bemore susceptile to the catabolic action of duodenaljuice.The patient presenting the extreme fall in jejunal
lactase (Fig. 4) is an example of an individualvariation. For unknown reasons the jejunal entero-cytes in this patient temporarily seem to have aselective decreased level of lactase compared withthe other enzymes.
Alterations in the amount of enzyme related toDNA reflect the fluctuations at a cellular level. This
study therefore presents evidence for the fact thatthe increased amount of enterocytes shown inmorphological studies are more or less deficient inenzymes. Also, when expressed per mg protein, thebrush border enzymes show a tendency to decreaseexcept for lactase and sucrase in the postoperativeileum. Whether the total amount of brush borderenzymes per unit length intestine is changed post-operatively cannot be answered in this study.However, in experimental studies in rats it has beenshown that, even if the enzymes decrease per mgprotein and, even more pronouncedly, per mg DNAafter jejunoileal shunt operation the amount of theseenzymes per unit length intestine is increased.7This may also be true for the human.
In the present study we have also shown that,except for maltase, there is a close correspondancebetween the amount of immunoreactive enzymeprotein and enzymatic activity before and after by-pass operation. Thus we conclude that the decreasedenzymatic activity is not caused by production ofimmunoreactive enzyme molecules with zero ordecreased enzymatic activity. Provided that thesolubilisation procedure also releases possibleenzyme precursor molecules from intracellularparticles we can also conclude that the suggested,increased population of immature cells on the villidoes not contain an increased amount of immuno-reactive enzyme precursors. Where maltase is con-cerned the precipitate area expresses directly theamount of this enzyme, while the enzymatic measure-ment represents the activities of both maltase andthe sucrase-isomaltase complex. Thus the immuno-electrophoretic measurement of this enzyme was notexpected to be correlated with the enzymatic activity.
The jejunoileal shunt operations were performed atsurgical department D, Herlev Hospital, Denmark,as a part of the Danish Obesity Project. The authorsare grateful to Ms D Anthonsen, Ms D Rasmussen,Ms M v Holstein, and the nursing staff of medical-gastroenterological Department C, Herlev Hospital,for skilful technical assistance. This work was sup-ported by the Danish Medical Research Council(No. 512-10175), P Carl Petersens Fond (No. B1076) and the Novo Foundation.
References
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668 Skovbjerg, Gudmand-Hoyer, Noren, and Sjostrom
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