Functional histochemistry of small bowel mucosa in ...Functional histochemistry ofthe...

Gut, 1964, 5, 145

Functional histochemistry of the small bowel mucosain malabsorptive syndromes

H. M. SPIRO', M. I. FILIPE, J. S. STEWART, C. C. BOOTH, AND A. G. E. PEARSE

From the Departments ofPathology and Medicine, Postgraduate Medical School,University ofLondon

EDITORIAL SYNOPSIS This study shows that in the flat mucosa of the small intestine in patients withidiopathic steatorrhoea many enzymes are lacking in normal activity but the histochemical reactionstend to be stronger in those patients who have had a gluten-free diet.

The flat mucosa of the small bowel in idiopathicsteatorrhoea lacks the normal activity of manyenzyme systems. Biochemical studies have shownthat such functions as the splitting of disaccharides(Santini, Aviles, and Sheehy, 1960) or the esteri-fying of palmitate (Dawson and Isselbacher, 1960)are impaired. A histochemical survey (Padykula,Strauss, Ladman, and Gardner, 1961) has indicatedthat in general the activity of esterase, adenosinetriphosphatase, and succinic dehydrogenase in theintestinal mucosa is diminished.

After a patient with idiopathic steatorrhoea takesa gluten-free diet, the mucosa of the small boweloften becomes less flat. The relation of enzymeabnormalities to the degree of intestinal mucosaldamage has not yet been established. This studyreports a histochemical survey of the jejunum innormal subjects, in patients with untreated idiopathicsteatorrhoea, and in patients with a variety of otherabsorptive disorders. In addition, studies have alsobeen carried out in a number of patients withidiopathic steatorrhoea treated with a gluten-freediet.

METHODS

The biopsies of the intestinal mucosa were obtained withthe intestinal biopsy capsule (Crosby and Kugler, 1957),the site of each biopsy having first been confirmedradiologically. The specimen was immediately laid outand cut into three parts: one for photography and routinehistology, one for study with the electron microscope,and one for histochemical studies. This last portion waslaid between two pieces of fresh liver for support, theliver serving also as a control for the validity of the

'Senior scholar of the American Cancer Society, 1962-63, in receiptof a grant for support of this study. Present address: Yale Universi.ySchool of Medicine, New Haven, Conn., U.S.A.

enzyme methods. It was then frozen in liquid air and wasstored in an airtight polyethylene bag at - 70°C.

Sections from each biopsy were cut at 8,u in a cryostatat - 18°C., picked up on coverslips, air dried, and thensubjected to the appropriate histochemical determination(Pearse, 1960). At least two sections of each biopsy werestudied, and many of the specimens were studied onseveral occasions. Usually, a section from a normalcontrol was studied with each specimen from a patientwith idiopathic steatorrhoea. Whenever more than onespecimen from a subject was studied, both were incubatedsimultaneously in the same coplin jar in order to ensureuniformity of conditions. Because of the possibility thatstorage might lead to a decrease in enzyme activity, anormal biopsy specimen as old as the oldest biopsy froma patient with idiopathic steatorrhoea studied was pro-cessed in order to indicate any decreased staining.The following histochemical determinations were

carried out: Alkaline phosphatase (Burstone, 1958a), acidphosphatase (Burstone, 1958b), non-specific esterase(Holt, 1958), cathepsin (Hess and Pearse, 1958), glucose-6-phosphate dehydrogenase (Hess, Scarpelli, and Pearse,1958), monoamine oxidase (Glenner, Burtner, and Brown,1957),D.P.N.-and T.P.N.H.-diaphorase (Hess etal.. 1958),succinate dehydrogenase (Hess et al., 1958), and leucineaminopeptidase (Nachlas, Crawford, and Seligman, 1957).Some specimens were also studied foractivity of ,B-glucosa-minidase(Pugh and Walker, 1961), and for ,-glucuronidase(Seligman, Tsou, Rutenburg, and Cohen, 1954) and fora-glycerophosphate dehydrogenase (Hess et al., 1958).Activity was graded as from 1 to 4, the reaction of thenormal specimen being set at 4. Such numerical valueswere not intended to be exact but rather to serve as aconvenient index of qualitative change only; they refer tothe intensity of the reaction, not to its distribution. Thevalue of 4 is normal, of 3 less so, while 2 and 1 indicatedefinite degrees of decrease in enzyme. Other sectionswere stained with haematoxylin and eosin; the epithelialheight was independently graded by three observers fromnormal (4) to very flat (1). Another portion of thespecimen was studied by the dissecting microscope,

145

on October 25, 2020 by guest. P

rotected by copyright.http://gut.bm

j.com/

Gut: first published as 10.1136/gut.5.2.145 on 1 A

pril 1964. Dow

nloaded from

http://gut.bmj.com/

H. M. Spiro, M. I. Filipe, J. S. Stewart, C. C. Booth, and A. G. E. Pearse

classified by the scheme of Doniach and Shiner (1957),and photographed. All slides were reviewed by at leasttwo observers, one of whom knew nothing about thepatients.

SUBJECTS

The subjects studied were 19 control hospital patientswith a variety of diagnoses such as chronic pyelonephritis,diverticulitis, etc., all without evidence of malabsorption;13 patients with idiopathic steatorrhoea none of whomwas taking the gluten-free diet, and in five of them asecond biopsy was carried out after they had received agluten-free diet. Four other patients who were alreadytaking a gluten-free diet also underwent biopsy. Eightother patients with other forms of malabsorption werealso subjected to biopsy.

RESULTS

CONTROL SUBJECTS The control subjects all showednormal finger-like vili with a few leaf-like forms.Since their histological and histochemical findingswere likewise normal, these are omitted from theTables, but some representative sections are presentedin the Figures.The reaction product which reflects succinate

dehydrogenase activity of the mitochrondria isdeposited sharply and clearly in the supranuclearportion of the surface epithelial cells in the form ofblack dots. There is evidence of weak activity in theinfranuclear zone as well. Equally prominentdeposits, and therefore equally strong activity, arenoted along the entire length of the villous epi-thelium, with a slight lessening of activity towardsthe bases of the villi. In the crypts, succinatedehydrogenase activity is present in lesser amountsthan in the villous or surface epithelium. Glucose-6-phosphate dehydrogenase and ax-glycerophosphatedehydrogenase have a distribution which parallelsthat of succinide dehydrogenase but sometimessome cells in the crypts react quite strongly. Theactivity of D.P.N.-diaphorase and T.P.N.-diaphoraseis similar to that of succinate dehydrogenase but isfound in much lesser amounts in the crypts (Fig. 1).

Alkaline phosphatase activity is sharply andclearly defined as a deposit overlying only the brushborder of the surface epithelium. Its activitydisappears at the junction of the villus with thecrypts, but in the lamina propria isolated areas ofactivity can be seen in occasional cells and in somemacrophages. Acid phosphatase activity is located inthe supranuclear zone of the surface epithelium.Activity decreases slightly towards the bottom of thevilli and is absent in the crypts. Many macrophagesin the lamina propria stain strongly by the azo dyemethod used and occasionally some activity is seenin the crypt cells (Fig. 2).

Leucine aminopeptidase activity is demonstratedby staining of the outer border of the villous andsurface epithelial cells. Evidence of activity abruptlydisappears at the junction of the villi with the crypts,and no activity is seen in the crypt cells. One normalcontrol showed diminished leucine aminopeptidaseactivity.

Non-specific esterase activity is strongest in thecells of the surface and villous epithelium. Theenzyme reaction product is supranuclear in location,but situated beneath the brush border, and isstrikingly uniform in definition. Activity diminishes,but does not disappear, at the crypt area. Theargentaffine cells also show strong esterase activity(Fig. 3). There is very slight activity also in themuscularis mucosae. Macrophages in the laminapropria sometimes show strong esterase activity.Monoamine oxidase activity is concentrated in the

supranuclear portion of the surface epithelial cells,with some activity evident below the nucleus.Activity is strongest in the tips and sides of the villi.Usually the enzyme product is a dense blackparticle, but sometimes the half-reduction product ofthe tetra-nitro-BT is found as a soft brown pre-cipitate.

/3-Glucosaminidase activity occurs in the form ofsmall granules localized just beneath the apicalsurface of the villi and in macrophages of thelamina propria. /3-Glucuronidase activity is foundpredominately in the surface epithelial cells ofthe villi.

UNTREATED PATIENTS WITH IDIOPATHIC STEATORRHOEAThe results in these subjects are presented in Table I.There was an overall relationship between the degreeof mucosal damage and the extent of the histo-chemical abnormalities.

In the mucosa of the jejunum in these subjects, thegeneral decrease in succinate dehydrogenase activitywas most marked in the surface epithelial cells andin the cells which line the luminal surface of thevilli. In eight of these patients activity was signifi-cantly decreased and in three less so. In one patientsuccinate dehydrogenase activity was normal.Activity in the crypts was not always decreased asmuch as that in the surface epithelium.The activity ofglucose-6-phosphate dehydrogenase

in the idiopathic steatorrhoea bowel was decreasedin all untreated patients in whom it was studied, butwas significantly low in seven of nine subjects. In 11of 13 subjects the activity of D.P.N.- diaphorase wasdecreased, while in nine of 12 subjects, T.P.N.-diaphorase activity was less than normal (Figs. 4aand b). ox-Glycerophosphate dehydrogenase activitywas decreased in four of eight subjects only.

In none ofthe patients with idiopathic steatorrhoeawere gross abnormalities of alkaline phosphatase

146



j.com/


pril 1964. Dow

nloaded from

http://gut.bmj.com/

Functional histochemistry of the small bowel mucosa in malabsorptive syndromes

FIGF. .FIC;. 1 ci11_l _ S | | | 5 | | I[G. 2

FIG. 3

FIG. 1. Normal jejunum. T.P.N.H.-diaphorase. x 100.The reaction is strongest in the supranuclear portion of thesurface epithelial cells. The reaction for succinic dehydro-genase is similar, but is somewhat stronger in the cryptcells.

FIG. 2. Normal jejunum. Acid phosphatase. x 180. Thesupranuclear zone of the surface epithelial cells containsmost of the reaction product, but some macrophages in thelamina propria are also strongly reactive. The brushborder does not contain the product of enzyme activity.

FIG. 3. Normal jejunum. Non-specific esterase. x 370.The argentaffin cells show a strong enzymic activity.

FIG. 4a. Jejunum, idiopathic steatorrhoea, untreated.D.P.N.H.- diaphorase. x 100. The weak- activityrepresent-ed by the faint deposits should be compared with the strongactivity in the villifrom a normal subject (Fig. 4b x 100).The sections were incubated together for the same lengthoftime.

FIG. 4a

FIG. 4b

147



j.com/


pril 1964. Dow

nloaded from

http://gut.bmj.com/


FIG.

9 'FIG. 5. Jejunum, idiopathic steatorrhoea, untreated. Alka-line phosphatase. x 100. Although the villi have fused, thectivity of alkaline phosphatase is strong in the brush

border of the flattened surface.

FIG. 5

.....

- | l | ll|FlG6a. Jejunum, idiopathicsteatorrhoea, untreated. Acidphosphatase x 360. The spotty

~~ ~~ repatre of the deost

ty can be produced in*~ ~ .. normal epithelium by gross

.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~Q

uinder-incubation, and probably~~~~represents the precise local-

ization of acid phosphatase in....X ..F..thepatient with idiopathic

4 steatorrhoea. It may be taken toindicate a lesser degree ofactivity as compared with the

__.. more diffuse reaction in a normal

E_ _,.. -( also Fig. 6b x 370). See

6a F F X.inreseinhigt aksFIG.65b

' .,. e . ... ... ......i':|. ;§ a,:. <* .. inthe~~~~~~~~~~~~~~~~~~~~~~~~~... .............. .....

Fig. 7a. Jejunum, idiopathicsteatorrhoea, untreated.Haematoxylin and eosin.x 370. The surface epithelialcells in the untreated patientare short and contrast with theincreased height of cells fromthe surface epithelium of thesame patient six weeks afterthe start of the gluten-free diet(Fig. 7b x 370). Most of theincrease in height takes placein the supranuclear zone, thearea of maximal concentrationof most enzymes.

FIG. 7b

148

ICi;. 7a1



j.com/


pril 1964. Dow

nloaded from

http://gut.bmj.com/


FIG. 8a FrG. 8b FIG. 8c

FIG. 8a. Jejunum, idiopathic steatorrhoea, untreated. Acid phosphatase. x 100. The weak reaction in the untreatedpatient should be contrasted with that from the same patient.four weeks later (Fig. 8b x 100). The cells are taller andenzymic activity has increased in the supranuclear zone. A tissue sectionfrom a normal subject is shown in Fig. 8c ( x 100)to emphasize that while enzymic activity returns towards normal a short time after the start of the gluten-free diet, it doesnot yet return completely to normal.

FIG. 9a. Jejunum, idiopathicsteatorrhoea, untreated.Monoamine oxidase x 100.The activity in the surfaceepithelial cells of the untreatedpatient contrasts with thesharper and blacker deposits in abiopsy from the same patientsix weeks after starting therapywith the gluten-free diet(Fig. 9b x 100).

FIG. 9a FIG. 9b

activity observed (Fig. 5). The shortening or fusingof villi led to an overall activity which was less thannormal, but the extent was diminished only by thedecrease in surface area. Activity over the surface ofthe bowel continued strong, though rarely dyedeposits were spotty and sparse. Acid phosphataseactivity was markedly decreased in seven of 12patients; in several, although the intensity of theactivity in the cytoplasm of the surface epitheliumwas not decreased, areas of acid phosphatase activity,presumably corresponding to regions with a high

lysosome content, were spread all over the cytoplasminstead of being localized mainly just under thebrush border. In some areas in all subjects withidiopathic steatorrhoea the reaction in the brushborder was spotty (Figs. 6a and b and Fig. 8). Evenwhen acid phosphatase activity was low in the surfacecells, it was still strong in the macrophages of thelamina propria.Of 13 patients. leucine aminopeptidase activity

was decreased in three. Esterase activity variedmarkedly; 10 of 12 subjects generally showed a

149



j.com/


pril 1964. Dow

nloaded from

http://gut.bmj.com/

H. M. Spiro, M. L. Filipe, J. S. Stewart, C. C. Booth, and A. G. E. Pearse

lessened activity in the epithelium but in only sixwas the reaction as weak as 1-2. In several patients,although overall esterase activity was markedlydecreased, esterase activity was strong in themacrophages of the lamina propria. Monoamineoxidase activity was moderately decreased inseven of eight patients (Fig. 9a); 3-glucosaminidaseactivity was also very low in the subjects studied, butin one case there was a great increase in the activityin macrophages.

TREATED SUBJECTS WITH IDIOPATHIC STEATORRHOEAThe overall enzymic reactions in patients studiedduring therapy with a gluten-free diet are summarizedin Table I. After therapy, only one patient showed acompletely normal histochemical reaction. Theresults in five patients in whom biopsies wereobtained before as well as during the time that theywere taking the gluten-free diet are shown in Table II.It will be noted that in three of five patients in whombiopsies were obtained before and one to ninemonths after they began to take the gluten-free diet,there was an increase in the height of the surfaceepithelial cells (Figs. 7a and b). In these patientsthere was a corresponding increase in the histo-chemical activity of many enzymes (Figs. 8a and band Figs. 9a and b), but this was not always an exactcorrelation: in subject 1, for example, succinatedehydrogenase activity remained unchanged despitea marked increase in the height of surface cells. Itshould be emphasized that no change in alkalinephosphatase activity was noted, except for thatbrought about by improvement in the villousarchitecture, and in other treated patients alkalinephosphatase activity was also normal.

TABLE IRESULTS OF HISTOCHEMICAL REACTIONS IN PATIENTS WITHIDIOPATHIC STEATORRHOEA BEFORE AND DURING A GLUTEN-

FREE DIET

Untreated Idiopathic Treated IdiopathicSteatorrhoea Steatorrhoea

1-2 3-4 1-2 3-4

No. No. No. No.

Alkaline phosphataseLeucine amino peptidaseEsteraseCathepsinAcid phosphataseSuccinate dehydrogenaseT.P.N.-diaphoraseD.P.N.-diaphoraseGlucose-6-phosphatasea-GlycerophosphataseMonoamine oxidaseP-GlucosaminidaseO-GlucuronidaseLactate dehydrogenase

0 10 (100%) 0 8 (100%)3 (23%) 10 (77%) 0 7(100%)6 (50%) 6 (60%) 2(25%) 6 (75%)3 (600%) 2 (40%)- - - -

7 (58%) 5 (42%) 3 (38%) 5 (62%)8 (67%) 4 (33%) 2(22%) 7 (78%)9 (75%) 3 (25%) 1 (17%) 5 (83%)11 (85%) 2 (15%) 4(44%) 5 (56%)7 (78%) 2 (22%) 1 (20%) 4 (80%)4 (50%) 4 (50%)- - - -7 (78%) 1 (13%) 3(37%) 5 (63%)7 (100%) 0 -

7 (100%) 0 - - -8 (100%) 0 - - - -

OTHER FORMS OF MALABSORPTION None of thesesubjects had a flat jejunal mucosa. The villi in twosubjects with pancreatic steatorrhoea and in one witha subtotal gastrectomy and in one with idiopathicosteomalacia, were leaf-shaped rather than filiform,and in these specimens the activity of oxidoreductaseenzymes was slightly reduced. Two subjects withtropical sprue both showed partial villous atrophyand the activity of enzyme systems in these biopsieswas generally slightly, but not profoundly decreased.

In the two subjects with Whipple's disease, thesurface epithelium showed normal enzymaticactivity when the specimen was incubated for anormal period of time. In addition, there was an

LE IISOME HISTOCHEMICAL REACTIONS IN PATIENTS STUDIED BEFORE AND DURING GLUTEN-FREE DIET

Therapy Epithelial Cell Phosphatase Esterase Leucine Amino- Monoamine Diaphorase SuccinateHeight peptidase Oxidase Dehydrogenase

Alkaline Acid D.P.N. T.P.N.

4 2 34 3 4

4 3 24 2 3

4 1 14 3 4

- 1 24 3 3

4 4 44 3 3

23

44

44

3

24

22

3

3

1 2 12 3 2

1 2 12 3 3

1 2 14 4 1

1 1 13 3 3

3 3 42 3 3

GF=Gluten free

23

22

24

2

4

33

FAA GF

6 wk.

FAB GF

3 mth.

FAC GF

1 mth.

FAD GF

9 mth.

E GF6 mth.

FA=Folic acid

150



j.com/


pril 1964. Dow

nloaded from

http://gut.bmj.com/


l

FIG. I Oa FIG. I ObFIG. 10. Jejunum. Whippls's disease. Acidphosphatase. x 140. (Fig. 10a x 100). Non-specific esterase x 140 (Fig. JOb).A short incubation period was necessitated by a strong macrophage activity and has produced an interrupted appearancein the deposits representing acid phosphatase activity in the surface epithelium cells. The surface epithelium has, however,normal enzymic activity, but the intense enzymic reactions in the niacrophages of the lamina propria which also stainpositively with P.A.S.

intense reaction of the P.A.S.-positive macrophagesof the villi towards most of the enzymes studied(Figs. lOa and b). Acid phosphatase, non-specificesterases, /3-glucosaminidase, and 3-glucuronidasewere especially active. The dehydrogenases anddiaphorases were present, but weak, in these cells.

DISCUSSION

In the epithelial cells of the normal jejunum,enzymatic activity is greatest at the tips of the villi,decreasing towards the junction of the villi with thecrypts. Cells are formed in the crypts and as theymigrate towards the tips of the villi they acquirelarger amounts of enzymes, presumably as areflection of the increase in their absorptive andmetabolic capacities (Padykula, 1962).

In the mucosa of patients with idiopathic steator-rhoea whose jejunal mucosa was flat enzymaticactivity was generally reduced; less marked changeswere present in patients with partial villousatrophy. In this study the oxidative enzymes,notably succinate dehydrogenase and glucose-6-phosphate dehydrogenase and monoamine oxidase,were most consistently depressed, along with T.P.N.-and D.P.N.-diaphorase. Non-specific esteraseactivity and that of acid phosphatase and /3-glucos-aminidase were often reduced. Leucine animo-peptidase was normal in three of 14 patients.An exception to this general lowering of enzyme

activity was the normal activity of alkaline phospha-6

tase, which contrasts with the atrophy of themicrovilli seen in electron micrographs. It must bepresumed that the microvilli which persist provideenough enzyme to yield a normal appearing reactionby light microscopy. These findings accord with thoseof Padykula et al. (1961) but not with those of Bolt,Pollard, and McCool (1960). Review of Bolt'spublished photographs, however, suggests that thereaction is of normal intensity over the surfaceepithelium and that it is decreased in extent only byshortening and fusing of the villi. Plosscowe, Berg,and Segal (1963) have recently recorded normaltrimetaphosphate and alkaline phosphatase activityin the epithelium of the bowel in idiopathic steator-rhoea.While the present study confirms and extends the

original observations of Padykula and her colleagues(1961) of the decrease of enzymatic activity in theidiopathic steatorrhoeal bowel, it did not produceevidence to support their conclusion that in idio-pathic steatorrhoea the surface epithelium alone isdepleted of enzymes and that the sides of the villimaintain their normal enzymatic reaction.Not all patients with idiopathic steatorrhoea show

a reduction in enzyme content, but neither is thehistological abnormality in idiopathic steatorrhoeaspecific: it can be mimicked by the effects ofanoxemia in animals, or of such diseases as regionalenteritis, and presumably of other injuries as well.For this reason it seems likely that what is termedidiopathic steatorrhoea comprises the end-result of

15I



j.com/


pril 1964. Dow

nloaded from

http://gut.bmj.com/


several pathological processes, not all of which mayhave the same enzyme defect as that of idiopathicsteatorrhoea. Further, symptoms of idiopathicsteatorrhoea may improve or worsen without relationto dietary gluten, and this may well be mirrored invariations in mucosal enzymatic reactions. Forexample, in this study two patients with idiopathicsteatorrhoea who were not taking a gluten-free dietshowed many normal histochemical reactions: thesteroids which one subject was taking at the time ofbiopsy may have been contributory. The otherpatient was asymptomatic without therapy; absorp-tive studies were normal, except that for xylose, yetthe mucosa of the small bowel was flat with epi-thelial cells of subnormal height. The many normalhistochemical reactions of the small bowel mucosapresumably reflect the good absorptive capacity(Fig. 11).The results of the study indicate that folic acid,

which many of the subjects had been taking for longperiods, had no effect on the appearance of theenzyme content of the mucosa of the small bowel.

In the patients studied after they had begun totake a diet free of gluten, a general increase inenzymic activity in the mucosa was noted, but notall patients who took a gluten-free diet showed anyincrease in enzyme activity, regardless of clinicalimprovement. This may have several explanations:the patients may be gluten-sensitive, their flatmucosa representing a non-specific response toinjury; repair of the enzyme defect in gluten-sensitive subjects may lag behind the clinicalimprovement; or the disease may have been of suchseverity as to prevent reconstitution of normalenzyme systems. Long-term studies of histochemicalfindings in relation not only to biochemical abnormal-ities but also to the clinical response may help toresolve the discrepancy noted by others betweenclinical response and histological improvement.

It is also possible that variation in histochemicalresults represents only sampling error. The mucosallesion of sprue may be spotty, a fact noted byDoniach and Shiner (1957) and by Rubin and hishost (1960), but often not taken into account byothers. In the same biopsy specimen (Fig. 12) may beobserved areas of partial villous atrophy adjacent toareas of flat mucosa; when very small pieces oftissue are studied, chance selection may play a role.Also, the biopsies before treatment were necessarilyolder than those during treatment; although anormal biopsy of comparable age was processedwith these specimens, the possibility remains thatdiminution in activity in a normal section might beless apparent than in one which has already lostsome of its enzyme activity. Finally, although aneffort was made to secure each biopsy from roughly

FIG. 1l. Jejunum. Idiopathic steatorrhea, untreated. Non-specific esterase. The mucosa throughout the specimen wasflatbuthistochemicalreactions, ashere, were intenselystrong.

FIG. ILbFIG. 12a. Jejunum, idiopathic steatorrhea, untreated.Haematoxylin and eosin. x 100. The mucosa shown in Fig.12a is flat, in contrast to that in Fig. 12b, which showspartial villous atrophy. Yet both were obtained from thesame biopsy section, to emphasize the occasionally patchynature of the idiopathic steatorrhoea lesion. The surfaceepithelium in Fig. 12a is taller than that in Fig. 12b andmight on that account alone be expected to show strongerenzymic reactions.

152



j.com/


pril 1964. Dow

nloaded from

http://gut.bmj.com/

Functional histochemistry of the small bowel mucosa in malabsorptive syndromes 153

comparable areas, some of the variations fromspecimen to specimen could represent differences inthe site of biopsy, the more so as enzyme activityvaries to some extent with the absorptive function ofthe segment. Very little is known about variations inhistochemical reactions in the small bowel. Thereremains a final possibility that the enzyme changesnoted are adaptive in nature and secondary toimproved absorption.The striking enzymic activity of the macrophages

of Whipple's disease noted by us and by Fisher(1962) should not be construed as of aetiologicalimportance at present. Macrophages in generalshow great histochemical enzyme reactivity. Thenormal enzymatic activity of the surface epithelium,however, emphasizes that the absorptive defect inWhipple's disease differs from that in idiopathicsteatorrhoea. Steroid and antibiotic therapy for threemonths in one subject led to no change in theenzymic activity of the macrophages on repeatbiopsy.The study provides some suggestion that there is a

rough relation between the morphology of the villiand their histochemical reactions. There was also arelation between the height of the surface epithelialcells and their content of acid phosphatase, succinicdehydrogenase, and D.P.N.- and T.P.N.- diaphorasesignificant at the 5% level. The more normal the cellheight, the more likely it was to have a normalenzyme complement. Thus normal villi usually havenormal enzyme activity and abnormal villi oftenhave lesser amounts. The only form of malabsorp-tion in which there was a significant decrease inenzymic activity was the mucosa of idiopathicsteatorrhoea, regardless of whether it was flat orshowed partial villous atrophy. In tropical sprue,only a very modest decrease in activity was noted,while the leaf-like villi of the patients with pancreaticsteatorrhoea had enzyme reactions which were normalor only very slightly decreased. We have no evidencewhether the increase in enzyme content precedeshistological improvement, but biopsy tubes capableof yielding multiple pieces of tissue in a short timeshould provide materials for an answer. Tentatively,it seems likely that the enzyme defects thus far notedfollow injury to the bowel, and that as the epithelialcells increase in height, so does their content ofenzyme increase. An important exception remains,however, in one patient (Fig. 11) with apparentlynormal enzymic activity in a flat mucosa. Morestudies are needed of the status of patients withtropical sprue and other forms of malabsorption aswell as of the effects of cortisone and gluten-free dieton the enzyme content of the small bowel.

Finally, what meaning should be attached to thedecrease in enzymes in the mucosa of the small

bowel in idiopathic steatorrhoea? Obviously thereought to be some correlation with malabsorption;and lack of enzymes, whether primary or secondary,may well lead to absorptive difficulties. Thus adeficiency of acid phosphatase, an enzyme linkedwith pinocytosis, might reflect a lack of imbibitionby the small bowel, or the lower activity of mono-amine oxidase might be linked with absorption ofunaltered toxic amines. But such speculation needsbiochemical corroboration, the more so as we donot know the quantitative meaning of the histo-chemical defect. In any case, the crucial enzymedefect, one which will not be repaired by the gluten-free diet, has yet to be discovered.

SUMMARY

In the normal small bowel of control patients,histochemical reactions were normal. A significantreduction in the activity of many histochemicalreactions was noted in the epithelium of the smallbowel of most patients with idiopathic steatorrhoea.Two patients showed predominantly normal histo-chemical reactions: one was being treated withsteroids, the other had a flat mucosa, but was inremission clinically and biochemically. Oxidoreduct-ases were decreased most often, but peptidases werealso diminished. Normal alkaline phosphataseactivity remained a prominent exception in themucosa of patients with untreated idiopathicsteatorrhoea.

After the administration of a gluten-free diet,histochemical reactions in the small bowel tended tobe stronger, and this seemed to be related to theincrease in the height of the surface epithelial cells.The administration of folic acid was not followed byimprovement in enzymic reactions.

In other forms of malabsorption, there was nomarked reduction in enzymic activity, but leaf-likevilli tended to show a slight decrease in enzymicreactions. In Whipple's disease, the P.A.S.-positivemacrophages also showed strong enzymic reactions,while the normal reactions of the surface epitheliumconfirmed that the absorptive defect lay beyond thesurface epithelium.

Tentatively, it seems likely that the decrease inenzymic activity follows rather than precedes themorphological changes in the bowel; the enzymedefect responsible for the initial damage to the bowelin idiopathic steatorrhoea should not return towardsnormal after therapy.

We are grateful to Mr. W. Brackenbury for the photo-graphs.

REFERENCES

Bolt, R. J., Pollard, H. M., and McCool, S. (1960). Staining ofenzymes in mucosa of the small bowel, using a peroral biopsytube. Amer. J. clin. Path., 34, 43-49.



j.com/


pril 1964. Dow

nloaded from

http://gut.bmj.com/

154 H. M. Spiro, M. I. Filipe, J. S. Stewart, C. C. Booth, and A. G. E. Pearse

Burstone, M. S. (1958a). Histochemical comparison of NaphtholAS-phosphates for the demonstration of phosphatases. J. nat.Cance, Inst., 20, 601-615.

(1958b). Histochemical demonstration of acid phosphatases withNaphthol AS-phosphates. Ibid., 21, 523-539.

Crosby, W. H., and Kugler, H. W. (1957). Intraluminal biopsy of thesmall intestine: the intestinal biopsy capsule. Amer. J. dig. Dis.,2, 236-241.

Dawson, A. M., and Isselbacher, K. J. (1960). The esterification ofpalmitate- 1 -CA by homogenates of intestinal mucosa.J. clin. Invest., 39, 150-160.

Doniach, 1., and Shiner, M. (1957). Duodenal and jejunal biopsies.It. Histology. Gastroenterology, 33, 71-86.

Fisher, E. R. (1962). Whipple's disease. J. Amer. med. Ass., 181,396-403.

Glenner, G. G., Burtner, H. J., and Brown, G. W. Jr. (1957). Thehistochemical demonstration of monoamine oxidase activityby tetrazolium salts. J. Histochem. Cytochem., 5, 591-600.

Hess, R., and Pearse, A. G. E. (1958). The histochemistry of indoxyl-esterase of rat kidney with special reference to its cathepsin-likeactivity. Brit. J. exp. Path., 39, 292-299.Scarpelli, D. G., and Pearse, A. G. E. (1958). The cytochemicallocalization of oxidative enzymes. II. Pyridine nucleotide-linked dehydrogenases. J. biophys. biochem. Cytol., 4, 753-760.

Holt, S. J. (1958). Indigogenic staining methods for esterases. InGeneral Cytochemical Methods, edited by J. F. Danielli, vol. I,pp. 375-398. Academic Press, New York.

Nachlas, M. M., Crawford, D. T., and Seligman, A. M. (1957). Thehistochemical demonstration of leucine aminopeptidase.J. Histochem. Cytochem., 5, 264-278.

Padykula, H. A. (1962). Recent functional interpretations of intestinalmorphology. Fed. Proc., 21, 873-879.Strauss, E. W., Ladman, A. J., and Gardner, F. H. (1961).A morphologic and histochemical analysis of the humanjejunal epithelium in nontropical sprue. Gastroenterology, 40,735-765.

Pearse, A. G. E. (1960). Histochemistry, Theoretical and Applied.2nd ed. Churchill, London.

Plosscowe, R. P., Berg, G. G., and Segal, H. L. (1963). Enzymehistochemical studies of human gastric and jejunal biopsyspecimens in normal and disease states. Amer. J. dig. Dis.,8, 311-318.

Pugh, D., and Walker, P. G. (1961). The localization of N-acetyl-3-glucosaminidase in tissues. J. Histochem. Cytochem., 9,

242-250.Rubin, C. E., Brandborg, L. L., Phelps, P. C., Taylor, H. C. Jr.,

Murray, C. V., Stemler, T., Howry, C., and Volwiler, W. (1960).Studies of celiac disease. II. The apparent irreversibility of theproximal intestinal pathology in celiac disease. Gastroenterology,38, 517-535.

Santini, R., Jr., Aviles, J., and Sheehy, T. W. (1960). Sucrase activityin the intestinal mucosa of patients with sprue and normalsubjects. Amer. J. dig. Dis., 5, 1059-1062.

Seligman, A. M., Tsou, K. -C., Rutenburg, S. H., and Cohen, R. B.(1954). Histochemical demonstration of 3-d-glucuronidasewith a synthetic substrate. J. Histochem. Cytochem., 2, 209-229.



j.com/


pril 1964. Dow

nloaded from

http://gut.bmj.com/

Functional histochemistry of small bowel mucosa in ...Functional histochemistry ofthe...

Documents

Transcript of Functional histochemistry of small bowel mucosa in ...Functional histochemistry ofthe...