Histochemistry of - Guthistochemistry, coloncancer, colonadenoma. The surface mucous gel layer of the large intestine probably serves as a lubricant to protect the mucosa. Its study

Gut 1997; 40: 782-789

Histochemistry of the surface mucous gel layer ofthe human colon

K Matsuo, H Ota, T Akamatsu, A Sugiyama, T Katsuyama

Department ofSecondInternal Medicine andLaboratory Medicine,Shinshu UniversitySchool ofMedicine,3-1-1 Asahi,Matsumoto 390, JapanK Matsuo

Department ofLaboratory Medicine,Shinshu UniversitySchool ofMedicine,3-1-1 Asahi,Matsumoto 390, JapanH OtaT Katsuyama

Department ofEndoscopy,Shinshu UniversityHospital,3-1-1 Asahi,Matsumoto 390, JapanT Akamatsu

First Department ofSurgery, ShinshuUniversity School ofMedicine, 3-1-1 Asahi,Matsumoto 390, JapanA SugiyamaCorrespondence to:Dr Tsutomu Katsuyama,Department of LaboratoryMedicine,Shinshu University School ofMedicine,3-1-1 Asahi,Matsumoto 390, Japan.

Accepted for publication23 January 1997

AbstractBackground and aims-Histochemicalanalysis ofthe surface mucous gel layer ofthe human colon is difficult, as it dissolvesin fixatives. This study was undertaken toexplore the surface mucous gel layer on

the normal mucosa and neoplastic tissuesof the large intestine. In addition, thedistribution of different mucins secretedfrom goblet cells was studied with a seriesofhistochemical stains for mucins.Methods-Twenty four surgically resectedspecimens were fixed in Carnoy's solutionand embedded in paraffin. In four cases,

the surface mucous gel layer was alsostudied in frozen sections. Serial sectionswere stained by a battery ofhistochemicaltechniques characterising mucins.Results and conclusion-The surfacemucous gel layer consisted of the innerand outer layers. The first covered theluminal surface of the mucosa, consistedof mucins, and showed a vertical stripedpattern. The second overlaid the first,showed a lateral striped pattern, and was

contaminated with bacteria and othersubstances. Their thickness in paraffinsections varied considerably among thesites in the large intestine, but was thethickest in the rectum and measured 12-7(SEM 6.0) ,lm and 88-8 (SEM 801) ,umrespectively. Mucins forming the innerlayer were obviously derived from gobletcells underlying it.(Gut 1997; 40: 782-789)

Keywords: surface mucous gel layer, mucin,histochemistry, colon cancer, colon adenoma.

The surface mucous gel layer of the largeintestine probably serves as a lubricant to

protect the mucosa. Its study in ordinarymicroscopical preparations is hampered by itsloss during fixation. Stabilisation of the surfacemucous gel layer was attained by Ota andKatsuyama by fixing surgically removedstomach in cooled Carnoy's solution, clearingin xylene, embedding in paraffin wax, andsectioning.' The surface mucous gel layer waswell preserved. Some of us applied this tech-nique to study the surface mucous gel layer ofthe large intestine and showed that it consistedof two layers, an inner obliquely striped layerand an outer multilaminated layer.2 Thepresent study was undertaken to elucidate thedistribution of the surface mucous gel layer onnormal mucosa and neoplastic tissues of thelarge intestine. In addition, the distribution of

different mucins secreted from goblet cells wasstudied by using a series of histochemical stainsfor mucins.

Materials and methodsTwenty four samples of surgically removedhuman colon were used in this study. Thesematerials were obtained from 22 cases of coloncancer, one case of adenoma, and one case offamilial adenomatosis coli. Locations of thesespecimens were caecum (one), ascendingcolon (three), transverse colon (four), sigmoidcolon (eight), and rectum (eight). Immediatelyafter resection all materials were opened alongthe contralateral portion to the lesion. Withoutrinsing, the specimens were laid flat with themucosal surface up and pinned on cardboard.In 13 cases, 3% alcian blue solution in distilledwater was sprayed onto the mucosal surfacebefore immersing in fixative to investigatewhether the mucous gel layer was an artifactduring fixation. All specimens were immersedin Carnoy's solution (ethanol 6:acetic acid3:chloroform 1, v/v/v) for two hours at 4°C.They were then placed in 100% alcohol.Materials were sliced longitudinally at regularintervals of 5 mm width, cleared in xylene, andembedded in paraffin. After histopathologicalexamination, several blocks, which wereobtained from the cut end of the materials orincluded non-neoplastic mucosa as well asneoplastic tissues, were selected for histo-chemical staining. Serial paraffin sections of3 ,um thickness were prepared and stained toanalyse mucins in the surface mucous gel layer.The Table gives the histochemical stains usedand their histochemical relevance. Alkalinehydrolysis (1% potassium hydroxide for15 minutes at room temperature) was per-formed to remove 0-acetylated groups of8-0-acetylated N-acetylneuraminic acid (8-O-AcNeuAc).

In addition, the surface mucous gel layer wasexplored in frozen sections in four samples,including two of ascending colon, one oftransverse colon, and one of rectum. Thesematerials were removed because of coloncancer, and tissue sections were obtained frommacroscopically normal portions. Immediatelyafter removal, these sections were laid inplastic cases (Cryomold, Miles Laboratories,Naperville, IL, USA) and snap frozen in OCTembedding medium (Miles Laboratories,Naperville, IL, USA) by submersion in liquidnitrogen. Sections of 5 ,um thickness were cuton a cryostat and mounted on poly-L-lysinecoated slides (Muto Pure Chemicals, Tokyo,Japan), dried at room temperature, coated with

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Histochemistry of the surface mucous gel layer of the human colon

Histochemical staining: relevance and sources of reagents

Method Histochemical relevance Sources of reagents

Alcian blue pH 2-5-PAS (AB/PAS) To identify acid and neutral mucins. Alcian blue (Chroma, Koengen, Germany); basicfuchsin (Wako Pure Chemical, Osaka, Japan)

High iron dianiine/alcian blue To differentiate sulphated mucins N,N-dimethyl-m-phenylenediaminepH 2-5 (HID/AB)24 from non-sulphated sialomucins dihydrochloride (Kanto Chemicals, Tokyo,

Japan); N,N-dimethyl-p-phenylenediaminemonohydrochloride (Sigma, St Louis, MO,USA)

Periodic acid/sodium To identify sialic acid with an Periodic acid (Wako Pure Chemical, Osaka,borohydride/potassium 0-acylated side chain (8-0- Japan); sodium borohydride (Wako Purehydroxide-PAS (PA/SB/KOH/ acetyl-N-acetylneuraminic acid or Chemical, Osaka, Japan); potassium hydroxidePAS)25 26 8-O-AcNeuAc). Such a sialic acid (Wako Pure Chemical, Osaka, Japan)

is most abundant in goblet cells ofthe large intestine

Periodic acid-cold thionin To differentiate sialic acid with anSchiff-potassium hydroxide-PAS adjacent hydroxyl radical (blue)(PA/TS/KOH/pAS)27 28 from those without adjacent

hydroxyl radicals (8-O-AcNeuAc,magenta)

0 2% celloidin in 1:1 v/v ethanol-diethyl ether,and stained by haematoxylin and eosin (H andE), alcian blue/periodic acid Schiff (AB/PAS),high iron diamine/alcian blue (HID/AB),periodic acid/sodium borohydride/potassiumhydroxide/periodic acid Schiff (PA/SB/KOH/PAS), or periodic acid/cold thionine Schiff/potassium hydroxide/periodic acid Schiff (PA/TS/KOH/PAS).The thickness of the surface mucous gel

layer was obtained by measuring at 10randomly selected points in each section withan ocular scale. The thickness was defined asthe distance from the outermost layer of thesurface mucous gel to the luminal surface ofthe surface lining cells. The data are expressedas means (SEM).

Results

materials were scattered among villi. Inpreparations in which alcian blue solution wassprayed on before fixation, the luminal surfaceof the surface mucous gel layer was con-tinuously covered by this pigment.The surface mucous gel layer seemed to be

a continuous eosinophilic layer coating themucosal surface (Fig 1 (also Fig 5A)), althoughits thickness differed considerably among dif-ferent sites in the large intestine. This layer wasdifferentiated into an inner layer and an outerlayer (Fig 2 and inset). The inner layer wasconsistently attached to the apical surface ofthe covering epithelia facing the intestinallumen and was continuous with the intracryptmucus. An obliquely striped pattern was oftenevident (Fig 2 and inset), except in the caecum,where this layer appeared as a homogeneousthin band. The outer layer overlaid the inner

HISTOLOGY OF THE SURFACE MUCOUS GELLAYERIn H and E preparations of ileum, no surfacemucous gel layer was seen and only amorphous

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Figure 1: Surface mucous gel layer on normal mucosa, transitional mucosa, and carcinoma.The suirace mucous gel layer is thicker on the transitional mucosa (arrow head) than thaton the normal mucosa (arrow), but has almost disappeared on carcinoma tissues. H and E,originally X 9-6. Transverse colon.

-~~~~~rd-SFigure 2: Normal mucosa. Higher magnification of thesquare shown in Fig 1. H and E, originally x 126. Inset:Higher magnification ofFig 2. The surface mucous gellayer consists oftwo layers, the inner layer (open star) andthe outer layer (closed star). The arrow indicates theboundery layer. Originally x 613.

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Matsuo, Ota, Akamatsu, Sugiyama, Katsuyama

one, was less eosinophilic than the inner layer,and mostly showed a lateral striped pattern(Fig 2 and inset). The surface gel layer wasthickest in the rectum and thinnest in thecaecum and measured 31a1 (7-2) ,um (range26-0-36-3 ,um) in the caecum, 34-4 (8-9) ,um(range 26-8-44-1 jim) in the ascending colon,50 5 (14-0) ,um (range 38-3-45 jim) in thetransverse colon, 62-0 (31 -9) jim (range17-3-115 ,um) in the sigmoid colon, and 88-8(80-1) jim (range 46&8-284-5 jim) in therectum. Except in the ascending colon, theinner layer was also thicker in the distal colonand measured approximately 5-6 (02) ,um inthe caecum, 4-7 (1 4) ,um in the ascendingcolon, 7 0 (3 7) jim in the transverse colon,7-6 (3 4) jim in the sigmoid colon, and 12-7(6 0) jim in the rectum. Cellular debris, foodresidues, and bacilli were often seen sand-wiched between laminated arrays of the outerlayer. The surface mucous gel layer was thickeron the transitional mucosa but had almostdisappeared on the adenoma and carcinomatissues (Fig 1), as described below.

HISTOCHEMISTRY

CaecumIn AB/PAS preparations, the goblet cells andintracryptal mucus stained blue/purple. Theinner layer stained intensely. The outer layer,on the other hand, stained magenta with

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Figure 3: (A-C) Ascending colon preparedfrom serial sections. Prepared at the samemagnification as Fig 5. (A) Originally x26 and inset X383. With AB/PAS, the innerlayer stains irregularly blue, whereas the outer layer stains bluedpurple with a stratifiedarrangement. (B) Sulphated mucins predominate not only in intramucosal mucus but alsoin the surface mucous gel layer, where the inner layer stains mainlyfor sulphated mucin,whereas the outer layer shows multilaminated structures. HID/AB stain. (C) The PA/SB!KOHIPAS sequence shows weak reactivity, not only in the mucosa but also in the surfacemucous gel layer, which shows the multilaminated pattern. PAISBIKOHIPAS,counterstained with haematoxylin.

B

Figure 4: (A) (ABIPAS) and (B) (HID/AB) Highermagnification of the surface mucous gel layer ofascendingcolon. The surface coat of the surface lining cells persistsunder the inner layer. Originally X 613.

horizontal stripes of blue/purple. Staining withHID/AB showed two layered patterns in eachcrypt; the goblet cells lining the upper cryptand luminal surface stained predominantlyfor sulphated mucins, whereas those liningthe lower crypt stained for non-sulphatedsialomucin. The inner layer of surface mucousgel layer stained black, whereas the outer layerwas a greyish blue colour with black horizontalstripes. In preparations stained with PA/SB/KOH/PAS goblet cells lining the crypts showedalmost equal reactivities, as did the intracryptalmucus. The inner layer stained more intenselythan the outer layer.

Ascending colonWith AB/PAS staining, goblet cells and intra-cryptal mucus predominantly stained blue/purple throughout the crypts (Fig 3A). Thesurface mucous gel layer stained blue/purple,showing a laminated structure (Fig 3A inset).With HID/AB staining, sulphated mucins pre-dominated in the goblet cells and also were themajor component of the overlying surfacemucous gel layer (Fig 3B and inset). Multi-laminated structure of the outer layer was evi-dent by mucin staining. In PA/SB/KOH/PASpreparations, the goblet cells disclosed definitebut weak reactivity at all levels of the mucosa(Fig 3C). The surface mucous gel layer alsostained faintly (Fig 3C inset). Under the innerlayer, the surface coat of the lining cells stainedclearly with both AB/PAS and HID/AB. Thisfinding was consistently found throughout thecolon (Fig 4A and B).

Transverse colonThis region of the large intestine showedtransitional patterns from the ascending to thesigmoid colon. In AB/PAS preparations, thecolour of intramucosal goblet cells seemed toshow more alcianophilia than that in theascending colon, especially in the upper andmiddle portions of the crypts. The inner layer

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of the surface mucous gel layer stained blue/purple and the outer layer showed amultilaminated structure. In HID/AB prep-arations, the number of goblet cells containing

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non-sulphated sialomucins slightly increasedin the upper portion of the crypts, and non-sulphated sialomucins increased in the innerlayer, although those containing sulphatedmucins remained predominant. The outerlayer also showed a laminated structure in PA/SB/KOH/PAS preparations, goblet cells liningcrypts stained less than those in the ascendingcolon. The surface mucous gel layer alsostained faintly.

Sigmoid colonIn the preparations stained with AB/PAS,goblet cells stained purple/red. The inner layer

Fti stained intensely red, whereas the outer layerstained pale magenta with a lateral stripedpattern. In preparations stained with HID/AB,the number of goblet cells containing sul-

; ts+ phated mucins decreased compared with the(.; transverse colon, especially in the upper layer

of the mucosa. The inner layer and outer layerstained only faintly, and the outer layer showedmultilaminated structures of fine grey stripeson an almost colourless background. Inpreparations stained with PA/SB/KOH/PAS,both goblet cell mucins and the surfacemucous gel layer were stained with moderateintensity. For the surface mucous gel layer,the outer layer almost always exhibitedthe multilaminated structure consisting ofalternating PAS positive and PAS negativelayers.

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Figure 5: (A-D) Rectum preparedfrom serial sections. (A) The double layered structureconsisting of the inner and outer layers is evident. The luminal surface of the surface mucous

gel layer is continuously covered by alcian blue, which was sprayed before fixation (arrows).H and E. (B) The inner layer of the surface mucous gel layer stains red/purple butoccasionally shows intense alcianophilia, whereas the outer layer shows the multilaminatedpattern. AB/PAS. (C) The inner layer of the surface mucous gel layer mostly contains non-

sulphated sialomucins, whereas the outer layer shows the multilaminated pattern consistingof non-sulphated sialomucin rich layers and sulphated mucin rich layers. HID/AB. (D)Both the intramucosal mucus and the surface mucous gel layer stain lightly. PA/SB/KOHIPAS, counterstained with haematoxylin.

RectumWith AB/PAS staining, goblet cells liningcrypts and intracryptal mucins stained red/purple (Fig 5B). The inner layer stainedsimilarly but occasionally showed intensealcianophila. The outer layer, on the otherhand, showed alternating laminated structuresconsisting of blue/purple and blue layers (Fig5B inset). With HID/AB staining, goblet cellslining the upper crypts predominately stainedfor non-sulphated sialomucins, whereas thoselining the lower crypts stained for sulphatedmucins (Fig 5G). The inner layer containedmostly non-sulphated sialomucins, and theouter layer consisted of non-sulphatedsialomucins and sulphated mucins abut-ting each other (Fig 5C inset). With PA/SB/KOH/PAS staining, goblet cells and thesurface mucous gel layer stained as lightly asthose in the transverse colon (Fig 5D andinset).

Colon adenomaAdenomas were recognised in 16 lesions fromseven cases. There were two cases of tubulo-villous adenoma with moderate dysplasia, eightcases of tubular adenoma with mild dysplasia,and six with moderate dysplasia. In most casesexamined, the surface mucous gel layer was notidentified on the adenoma tissues, regardless ofthe abundance of neoplastic goblet cells (Fig6A and B). A very thin surface mucous gellayer was seen in three cases. In these cases,only the outer layer persisted.

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Figure 6: (A) (H and E) and (B) (ABIPAS) Adenoma inthe transverse colon preparedfrom serial sections. Thesurface mucous gel layer is not present on the adenomatissues, regardless of the abundance of neoplastic goblet cells.Arrows indicate aggregated alcian blue, which was sprayedbefore fixation. Originally x 192.

Colon cancer

Hardly any surface mucous gel layer overlaidcancerous tissues in most lesions, althoughmucins derived from neoplastic goblet cellsoccasionally adhered to the surface. Even inthe region where numerous goblet cell typecarcinoma cells lined the luminal surface, theinner layer was never found (Fig 7A and B).

So called "blue crypts" and the surface mucous gellayerSo called "blue crypts"3 were found in foursigmoid colons and one rectum. They occurredsingly or included several crypts but couldnot be differentiated morphologically fromordinary crypts. Goblet cells lining these cryptsand lining the mucosal surface character-istically stained blue/purple with AB/PAS (Fig8A) but stained more intensely with HID/AB

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Figure 7: (A) (H and E) and (B) (AB/PAS) Cancer oftransverse colon preparedfrom serial sections. Althoughmucins derivedfrom neoplastic goblet cells occasionallyadhere to the surface, the inner layer is absent, even wherenumerous goblet cell type carcinoma cells line the luminalsurface. Originally x 192.

than those of the surrounding mucosa (Fig8B). The goblet cells were unstained by PA/SB/KOH/PAS (Fig 8C) but stained blue with PA/TS/KOH/PAS (Fig 8D). The inner layer alsostained similarly as long as goblet cells liningthe mucosal surface exhibited similar properties(Fig 8A, B, C, and D). In the more peripheralregion, similar mucins were spread along theboundary layer between the inner and outerlayers (Fig 9A and B). Thin layers of similarmucins were occasionally seen in the outerlayer.

Effect of alkaline hydrolysis on AB/PAS andHID/AB stainingPrior alkaline hydrolysis with potassiumhydroxide greatly enhanced PAS reactivityof most goblet cells throughout the colon.A similar effect was confirmed for theiralcianophilia. This pretreatment also enhancedHID reactivity of mucins. The surface mucousgel layer also gained higher reactivities for thesestains. The effect of this pretreatment was mostevident in the sigmoid colon, although thesurface mucous gel layer still stained less thanthat of the rectum with HID/AB stain. Afterthis pretreatment, goblet cells and the surfacemucous gel layer remained unstained with PA/SB/KOH/PAS and stained homogeneouslyblue with PA/TS/KOH/PAS. The "bluecrypts" and their mucins secreted, on the otherhand, were not influenced by this pretreatmentand therefore became indistinguishable fromthe surrounding crypts.

In the four cases examined by frozensections, the surface mucous gel layer appearedas a continuous layer coating the mucosalsurface and consisted of an inner and an outerlayer (Fig 10A, B, and C). The inner layerclearly showed a vertical striped pattern whichreflected the mucus spouting from each gobletcell. The outer layer showed no particularpattern, and its luminal surface was not clear.The thickness of the inner layer was 19-6 (310)pum (range 17-3-23 ,um) (Fig 10A, B, and C).In the inner layer, bacteria were seen along thevertical stripes (Fig 1OA and B).

DiscussionThe present study showed that fixation inCarnoy's solution preserves the surfacemucous gel layer in ordinary paraffin sectionsand allows these to be analysed by histo-chemical techniques.

Several attempts have been made to observethe surface mucous gel layer of the gastro-intestinal tract.'5 Kerss et al explored it bypreparing unfixed thin sections of rat, frog, andhuman stomach7 and recently Pullan et alprepared sections of human colon andmeasured its thickness.'6 They also usedCarnoy's fixation to support the existence ofthe surface mucous gel layer of the colon. Asstated previously, sectioning of the surfacemucous gel layer by razor blades is not easybecause of its stickiness.' In addition, histo-chemical analysis of this layer is difficult as itdissolves in solution during staining. Ota and

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Katsuyama showed that Carnoy'a useful fixative to preserve t]paraffin blocks.' The surface muwas histochemically analysed by Isections, although there was obviof the mucus during fixation anof paraffin wax blocks.' 2

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s solution was The present study confirmed two layers inhe mucus in the surface mucous gel, the inner layer and theicous gel layer outer layer, as suggested previously.2 Thispreparing thin structure was consistent in all cases examinedious shrinkage but the question still remained whether theId preparation structure was an artificial one or not. Several

findings, however, indicate the presence of thispattern. Firstly, the inner and outer layers wereclearly identified in frozen sections, althoughthe inner frozen layers were thicker than theywere in paraffin sections. The luminal surfaceof the outer layers was not very clear in thefrozen sections but was obvious in the paraffinsections. Secondly, size, distribution, andmucin content of goblet cells in the tissuepreparations fixed in Carnoy's solution did notdiffer significantly from these features of gobletcells fixed in ordinary formalin. This findingindicates that abrupt mucin discharge did notoccur during fixation in Carnoy's solution.Thirdly, the inner layer was consistently absenton adenoma or carcinoma tissues whichcontained numerous goblet cell type neoplasticcells. Fourthly, the surface coat, which char-acterises the apical surface of covering epi-thelium including goblet cells, preserved wellunder the inner layer, suggesting no disruptionof the apical plasma membrane. Fifthly, the

e inner layer was not found in ileum, which isalso rich in goblet cells. Other support comesfrom our study on the human stomach. Gastricmucosa often showed intestinal mucosa whichcontained plenty ofwell developed goblet cells.In the stomach, however, we have never foundthe inner layer in the surface mucous gel layer.

J So called "blue crypts" provided the clue toexplore the origin of mucins in the surface

--<-= mucous gel layer. The term "blue crypt"- was coined by Kato et al because of the

characteristic reaction to PAJTS/KOH/%C'f'<.^ PAS.3 1718 Its relevance as a precancerousN lesion was contradicted by recent study,'8

however. The present study showed that goblet:= cells lining blue crypts lacked 8-0-AcNeuAc,

- a histochemical marker of the large intestine,as goblet cell mucins remained negative by PA!

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Figure & (A-D) So called "blue crypts" in the sigmoid colon preparedfrom serial sections.Originally x 96. (A) Goblet cells lining blue crypts and the inner layer which cover only theextent of these crypts stain blue/purple. AB/PAS. (B) They stain more intensely than cryptsof the surrounding mucosa. HID/AB. (C) They are entirely unstained by the PA/SB!KOHIPAS sequence (arrows). (D) With the PAITS/KOHIPAS sequence they stainintensely blue and, therefore, were called "blue crypts".

B'Figure 9: (A,B) Higher magnification offig 8 (A,D).Evidence that mucus derivedfrom "blue crypts" spreadsalong the boundary layer between the inner and outer layers(arrow heads) or spreads into the outer layer (arrows).Originally X192.

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Figure 10: (A-C) Preparedfrom frozen sections. (A) Themucosal surface is coated by the surface mucous gel layerconsisting oftwo layers. Haematoxylin and eosin.Ascending colon. Originally x 192. (B) At highermagnification, the vertical striped pattern of the inner layeris evident. Bacteria are seen along the vertical stripes.Originally X 613. (C) The inner and outer layers are alsoevident. HID/AB. Originally X 766.

SB/KOH/PAS and stained blue by PAITS/KOH/PAS and previous alkaline hydrolysis didnot alter histochemical reactivities. Mucinsderived from "blue crypts" formed the innerlayer which covered only the extent of8-0-AcNeuAc free goblet cells. These resultsclearly indicated that mucins in the inner layerare derived from the goblet cells directlyunderlying it.Mucins free from 8-0-AcNeuAc'7-20 pro-

vided a good marker to explore the distributionof mucins in the outer layer where thesemucins spread, forming a thin layer. The multi-laminated structure found in the outer layermost possibly reflects different physico-chemical properties of the different types ofmucins20 21 secreted simultaneously from thesame goblet cell or separately from the gobletcells lining the different levels of the crypts.The effect of alkaline hydrolysis was found

not only in the stains related to 8-0-AcNeuAcbut also in stains for acid groups, HID and AB.The alkaline hydrolysis used in this study was

relatively mild, and is known to cleave 0-acyl

groups on C-7,8, and 9 of sialic acid. Inaddition it might also cleave the ester linkagebetween acid groups and hydroxyl groups ofsugar moieties, resulting in increased HID andAB reactivities of acid mucins.

Histochemical staining suggested that themucins in the outer layer stained similarly togoblet cell mucins. They also suggested that atleast sugar moieties of mucins were maintainedin the outer layer, as, for example, 8-0-AcNeuAc occupies the non-reducing terminalportion of oligosaccharides, and its existencesuggested the persistence of saccharide chains.Various mucins derived from goblet cells didnot mix homogeneously in the surface mucousgel layer but rather stacked on each other andformed the laminated structure, as exemplifiedby 8-0-AcNeuAc free mucins spread along theboundary layer and in the outer layer.The surface mucous gel layer varied in

thickness in different regions of the colon. Thedescending order of thickness was rectum,sigmoid colon, transverse colon, ascendingcolon, and caecum. Pullan et al also reportedthat the the surface mucous gel layer wasthicker in the left colon and the thickest layerwas in the rectum.'6 The thickness in theirreport was about twice that of ours. Thedifference between the two studies reflects themethod used, because, as described previously,there was appreciable shrinkage in Carnoy'ssolution.' 2 It remains to be clarified whatfactors determine the thickness of this layer.The surface mucous gel layer probably

reduces friction between the mucosa andfaeces.2' In this study, the inner layer onlyoccasionally contained bacteria. Thus it mayalso play a part in inhibiting the directadherence of bacteria to colonic epithelia.22 23

We express appreciation to Professor Kendo Kiyosawa andEmeritus Professor Seiichi Furuta for their advice andencouragement; and Drs Jun Nakayama, Keiko Ishii, IkuoMatsuyama, Masayoshi Hayama, and Osamu Yamagami fortechnical assistance.

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Histochemistry of - Guthistochemistry, coloncancer, colonadenoma. The surface mucous gel layer of the large intestine probably serves as a lubricant to protect the mucosa. Its study