THE HISTOCHEMICAL DEMONSTRATION OF a--GLUCOSIDASE IN

MAMMALIAN TISSUES*

ALEXANDER M. RUTENBURU, JULIUS A. COLDBARC, SELMA H. RUTENBURG,

AN5) RUTH T. LANC

}amzns Laboratory for Surgical Research and Departments of Surgery, Beth Israel Hospital and
Harvard Medical School, Boston, ,-llass.

Received for publicsst ion Novemhier 20, 1959

The o’o!oritsietric (!etermnination of cu-D-glUcOSi- ‘[‘he sumhislrate soluslions was prepared by (lis-
(htse with the svnsthetic SUI)strate 6-bromo-2- sollving 6-blromo-2-nsaphthyl a-D-glticopyranosiole

tia1)hthiyl a-D-g!Ucopyratioside t was repOrte(! re- mi 2-methoxyethanol at a concentration of 1

mg/ml. This solustioln was then dilusted with 0.1 M
eenstl (3). The Preso’tit report do’als with (a) a
phosphate husffer (pH 6.3) to yield a final sub-
niethod for the histocisemica! (lemsionstration of
strate concentration of 0.1 mg/mI. Lower concens-
tlsis enzyme irs tissue sections, (h) a survey of
trations of sutlistrate resitlted in w-eaker staining.
a-glucosiolase activity ins tho’ tissues of several
At higher concentrations the suslistrate precipi-
tsiatiinsials, amiol (c) o’hiatsges its etiZytiTlati(’ activity lated out of the buiffer soluilion.
its rstts subjeo’ted to st:trvatiolti. For sinsultaneous coupling, the stabilized
tetrazomsiusnsi salt of diorthoansisidine, CI No).
sumETHOD
:37255, fas( hluie B, (0.07 mg/mI) was added to the
Ins (isis method 6-bromol-2-napht-hol liberated sumlistrate solution arid sections incumbated for 1-2
by enszynsatic hydrollysis wsts converted to) a houtrs at 26#{176}C. This solution was changed every
highly colored insoluble azo dye by exposusre to 10 miniustes to avoid the presence of decomposed
tetrazolized diorthoaniisidine. disizonsi uim salt and colnsequment backgrou nd stain-
a-G!ucosidase activity cams be demonsstrated ing. There:tfter, the sections were washed in dis-
by (a) a simultaneous coupling technic whereini lilleo! water and mounted wilh Kaiser’s glycero-
enzymatic hydrolysis was carried out in the pres- gel . Tissuies Processed by this method Showed an
ence of a stabilized diazonsium salt or (li) a post- intensse purplish-red granular deposition of azo
coupling technic whereims, at a pH optimal for dye locmtlized at the sites (If enzymatic activity.
etszymatic hydrolysis, the liberated naphthol Sect-ions incubated for 8 hours showed a more
was susfficiently insoluble ansol l)roteims bound to intense reaction with the same distrihuslion of
remain localized at or tse:tr the sites of enzymatic enizymatic activity. Parallel sections handled by
activity, ansd was sumlisequsently converted to an the post -coumpling technic yielded a more homoge-
even less soluble azo dye by exposusre of the see- neouts deeper blue stain at the same sites.
tiotss to the diazonsiusm salt-. For post-coupling, folrmalin fixed sections
Tissumes were olbtainseol from amsimals killed by were inicuhated in sumbstrate solution for 2-18
exsansguinsalion. After fixatiots iti 10#{176},-Iseumtral housrs at 26#{176}C.After the appropriate incubation
formntlits for 8-24 houirs, 3-4 sections (10-15 s) period the sections were w’ashed in distilled
were cutt (Ins the carbons (!ioxi(!e freezinsg micro- water, anid 2-5 sections placed in 5 ml of fast blue
tome and imicutbated in 5-10 ml susbstrate solution B (1-2 mg/mI) dissolved in NaHCO3 (0.5-1.0
at 26#{176}C. mg/nnl) soluition (pH 8.0). After I minuste the see-
Folrniali ni was more ssut isfstc ory I hsuns acetone liolnis were removed, w’ashed and moumnted.
or alcohol for fixatiomi. Although some inshibition The suspernatant was tested for free naphthol
(15-20, ) resuilted followimsg exposusre to formalin, by increasinig the pH to 8 with NaHCO3 and add-
sufficierst activity rensained to yield a ssutisfactory ing the diazoniium salt. With very active tissues,
staimi in the mo)re active tissuses. Addition of chlo- the supernsalanit- sholwed a light- pumrple color.
ral hydrate did nsot stlter the degree of itshihition This imsdicstted diffumsiolrs of the enzyme amid the
caumsed liv formalins fixation (1). experimen( was rel)entted w’ith shorter periods of
incullatiolni.
* This investigat ions was suspp(Irted by research
grsunsts from the Natiomial Camso’er Inistituste, Nut- Fresh frozens secliomis (4-6 ) cut ins the cryostat
liomsal Institutes of Hesulths, l)epartmenst of Health, were also salisfactolry. These coutld either be
I’luscation amid Welfare, Bethesda 14, Marylansd. floated as free sectiolmss in the suslistrate solustion
t 1)ajac Labora(ories, (‘heniio’aI l)ivision, The
or prefixed to) slides. The latter maneusver resuslted
l3ordens (ki., 500() Langdots St., Philstdelphia 24,
Pa. ins about 80 loss oif enizymsiatic activity. This

268
 HISTOCHEMICAL METHOD FOR a-D-GLUCOSI DASE 269

effect has been reported w’ith other enzymes (7). the kidney the cylol)lasm (If the 1-uhuslar epithelial
Fumrlhemmore, in order to avoid diffusion artefacts cells stained most intensely (Fig. 5, 6).
with fresh frozen sections shorter periods (If in- The distribumlions 1)sutlernl in the I-issues of other
cunbalioln were requsired. mammals (moumse, rstbhit-, olog and gusinest l)ig)
Time and Temperature: Formalin fixed! see- was (he same as ins (he rat , except that the adremsals
lions were incuihaled at 26#{176},37#{176}and 50#{176}Cfor (If the gusinea l)ig vere llositive.
1 to) 24 hours. With the simuillaneous coutplinsg In (It-her experimeists (he effect (If feeditsg unit!
technic wherein the suibstrate solution was st arvalion on a-glutcosidsuse activity at variouts
chaniged every 10 minuites, sections incuml)ated at levels of the gast-rointeslimstl tract was evstlustled.
26#{176}Cfor 1-2 housrs show-ed the best results. For At varioums intervals after esuting, ans(l after 24,
the p01st coupling technic 2-18 houtrs at room tem- 48 anal 120 houmrs OIf slarvalion, rats were killed
peratunre was necessary. An overnight inicuba(ion liv exsanguninalions afl(1 :ippropriate segments (If

(12-18 houtrs) was genserally satisfactory utlthoutgh the stomach and instestinse stained for a-glumcosi-
more active specimens or fresh frozen sections dase. The intenisily of reaction could he corre-
reoumired shorter periods of incumbalion. Al higher lated with the insgestioni of food and the sutlise-
temperatures the susbstrate hydrolyzed :tnd the (luient stages of oligestiois. Enzymatic activity ins
diazolni uim salt decomposed. the muncosa of the rat olu(I(lenutm an(l pro)ximstl
Comment : As with all histochemical met holds jejunum was greatest after eating. Within 30
for the demonstration (If soluible enzymes, the minsustes the duodenal miisscosa staine(l most
pr(Il)lem (If artefacts (luse to diffussion of the eli- intensely. Thereafter there was progressive butt
zyme, or produicts (If hydrolysis of the substrate, i nconsslant decrease O)f react ivi Iy . Whsereas some
wsts encountered (2, 6, 8, 9). Attempts to) itppress rats showed lower levels of suctivity, others sholwed
diffutsiois of the enzyme oust of sections by incusba- fl() chstnige. After 5 olstys (If s(utrval ioni (lIlly a
lions in suibstrale s(Ilut(ion made hypertonic with faintly positive reacliolmi was (lemonstrah)le in the
so)diumm chloride (2 M) were usnsstmccessfutl hecautse olitodenutrn of several rsits. Rust kidney showeol 150

(If inhibit-ion of enzymatic activity. Several oiui- change in enzynisttic activity sufter s(arvations or
nsones (1 ,2-niaphthoqusinone, menadione so(liunm ingestion of foool.
bisuslfate, 2-methyl-I ,4-naphthoqutinone) which
DISCUS5I(IN
activsuted fl-glucosidase (2) did not increase
a-glucosidase activity. With formalin fixed sec- The histocho’tiiical d(’msiolnstration of a-gluicosi-
tions diffussion artefacts were insigniiflcanit. The (lase has not l)eeii r(’polrt(’(l previously. The
smmusltamieolus cousphing technic with shorter
enzymatic hiyolro!ysis of 6-bromo-2-rsaphsthivl
periods of inscusbalion also largely eliminltled olif- a-D-gluco’rstnoIsid(’ o!etisotistrable hsistolo’isennsi-
fumsion. cttlly h’ the tiio’thiool hio’rein reported ins the

RESULTS
(IUO(!(’titil o’pithio’liustiiof the rat and mi hiutiisin
duodenal fluid (3) tinny be olue to maltase. The
The followinig tissues of the rat were exaniiiseol: hiteratusro’ is colnitrovo’rsial on the existo’isc(’ of
salivary gland, esophaguts, stomach (funduss amid
maltaso’s sl)O’CificItllY adItl)to’d to) the hydrolysis of
anit rutm), small intestine (variosts levels), lsurge
maltose am! 110) otho’r a-glucoside. Phso’nvl
inslesline, heart, lutng, spleen, liver, kidney, pan-
a-D-glU(Osiole has h)0’(’ti rO’l)o)rte(l to) he a substrate
creas, adrenal, bladder, prolslale, testis, epidi-
for maltase in ro’latively cruiole enzyme pr(’parst-
dymis, seminal vesicle, vas deferens, ovary and
tions from barlo’v malt, brewer’s yeast, .1 sper-
uterus.
reactive.
Only
In
the
the
kidney
small
and
intestine
small
the
intestine
reaction
were
was gums oryzae, or varioums bst(’to’ria (4). A partially
confined largely to the mutcosal layer arid was most purified pro’putration of o’(luin(’ serum nialtase
intense in the duiodenutm, with progressive (!iniiniu- hvolrolvzed l)hseIlY! a-D-gluooside as well as
ti(Ini toward the ileutm. The cytolplasm of the epi- m:-tltoso’ (5). Accurate definiition of the substrate-
thelial cells of the dusodenal mucosa stained most spo’cificitv of a-gluso’osiolstso’s awaits furtho’r l)ulrifi-
intensely (Figs. 1, 2), w’i(h perinuclear accets- esutioni of these enizvniies.
tual ion (If activity. Brutnner’s glands and elements
Colorimetrio’ assay of rstt tissue hsoniogetiato’s
(If the myenleric nerve plexus were moderately
revealed a-glucosiolase stctivity in a-Il organs
reactive. Nusclei and goblet cells did not ststins.
a-ssayeol (3). Flistoo’hsetiiicstlly, hiowevo’r, enzymatic
The proximal jejuniusnn showed a lower order of
a-gluicosidase activity than the dutodenusni (Fig. activity can be olensomsstrstted only in the miiusrosa

3). Reactivity decreased fusrther iii the distal of tho’ duodo’niunii anid its th(’ ro’nal tuhuiles by the
jejutnusm. The ileuinn and colons (Fig. 4) as well sts niethod hiereiti do’scribo’d. Tho’ ohifference in the
the esophagus amid! stomach were negative. Ins so’nsitivitv of the hist-ochemicstl am! oolorinietric
270 A. M. RUTENBUR( ET AL.

.T
f4’’

r
IT

- -
- - - - -
- smodetsuni. Simsissltamseoums , - _snid!ume. Imiio’msse gransular n ins the cytop_asm
of itsto’stinns! (‘pit lieu mis. X 750.
Ft n. 2. Rstt duooletssttss - Post osslsIIli nsg to’chsmsioiuse.Thie cytoplsusnss of t lie dIll hselissl o’ells of t lie olusodensal
tts ttcos:s shissvs :sts i tito’msso’ honssogetteouss stain for a-gluscosidase. XS(K).
l”mn . 1. Isst jej umtsssmss. l st 0s ssmlsli tsg S o’chtsique. Mod!(’rsste resurt iots its I hue nsucosal epit heli nina. X809.

l)t)(((l11t0’S issst\. hs (Isle, itS l)Stt’t, t(I (litliitsishseO! to llsirtial itsartivatiots (If enzviiie liv fortsialits
a(ro’ssihlilit-y to suibstrsote of a-glusrosidstse in fixsstiots. Furtlsertsiore, the failumre to olemnomsstrsute
f )t’tssalims-fixe( I s((t I( )tls (( )t5il) r(’( I to I lie soluslile emszvtsisstic su’tivitv appr(Ipri:stelv by t-lse lsisto-
(‘tszvtsso’ :tvail:thle its tissume extracts ats I, in psurt, (hso’tsii(stl t(’(hstsic its orgsttss vlsoIse tissus(’ extn’acts
 FIISTSJO’HEMI(’AL METHOI) FOR a-tI-(LtO’OISI IIASE 271

FIG. 4. L.t colon. Post- couspling technique. Negative reactiomi. X250.

Fin. 5. Rat kidney cortex. Post- coupling technique. Cytoplasm of (ullnslar epitheliutns stainss most
itstemssely. Cell nuclei, red 1)10)0(1 cells sinsd glomerutli were uttsslainsed. X400.
Fin. 6. Rstt- kidney. Post coupling teo’hmiique. Cyloplstsm sf Ilie proximsd atsd dis( us! d’otsvolusled sshules
niost- resuclive for a-gluscosidstse. Collectinsg (lucls were less reaclive. XM(X).

are nio!erato’lv activo’, e.g. l)rolststto’, liver, ais(! tusbules, vho’to’as coloritsiu’tticall- kidtsev was
pstsress, may be partly olue to diffuse dist-riliu- aboust twice sts suotivo’ as a (ross-sestiots (If

tiomi of the enzyme throuighioust theso’ organs. (luo(lo’nsum. Tlsis suggests a gro’ttto’r degree (If

Ltizvtsiatic activity is highly concemitrstted in the loo’ahiztttioti of o’nzvtiie, or great-o’r stoco’ssihility (If

muicossul cells of the dunodenium and in the svto- enizvtiie to) suh)strstte its dumolol(’nal epit-hselial cells.

plasm of tho’ renal tuibuslar epitho’!isu! co’lls, thero’liv The role of renal a-glusrosiolstso’ in sarliolsvdrate

po’rmitting histochemicstl (lo’monstratioti of a-glum- metabolism is riot knsowis. This enzyme lists silso
(Osiolsts(’ in theso’ organs. Histoohi(’mi(’ally dusoole- beers founso! reguslarlv its the urine of mars atsd
nial mumeosa staine(! nioro’ int(’nso’lv than n’o’tsstl otiio’r nsistiiitiials (usispsslilislsed (lata). The Isis-
272 A. M. RUTENBURG ET AL.

tochsetssiral fitsdimigs its tlse ourretst stusdy itidi- TIme o(sl(Irimet nc estimatioti of $-o-glucosid-
use. J. Biol. (‘hem. 195: 607-614, 1952.
oato’ tlsat a-glucosio!ase its thso’ small insto’stinie of 3. Com.im.#{176}n, J. A., Tsou, K. C., Itr-TENBu’RG,
tlse rat so’rves a digestivo’ futsetiots and may be S. H., RUTENBURG, A. M. ANtI SELnGMAN,
A. M.: A method for the colorimetric de-
ims(r(aseo! sulstptivelv ins responsso’ to the ingestion
terminlstli(Ins of a-D-gluiC(Isi(!stse with a chrom-
of foIoIu!. ogenic substrate. Arch. Biochens. Biophys. 75:
435-442, 1958.
SUMMARY 4. COTTSCHALK, A.: a-D-C!uscosidases. In i’he
Enzynses. Susmnser, J. B. arid Myrback, K.,
A lsistochsemsiical mo’tlsolsl for the (lemonsstrationi e(ls., Acad. Press, New- \ork, 1950.
5. LIEBERMAN, I., AND E’ro, W. H.: Pusrificalion
(If a-D-gluscOSidstSe is pro’sent(’( I. TIso’ procedUr(’
ssmsd properties (If equsitie serumm maltstse. J.
atsol the l)rol)lo’nsls onscolunmstereol its its o!evelopnient Biol. (‘hem. 225: 899-908, 1957.
:tro’ o!iscusso’o!. Surveys of iiiamtiist!ian tissues 6. RUTENBLRG, A. M., COHEN, R. B. AND SELIG-
MAN, A. M.: A niew methold for the hislo-
shsowe! that this enzyme is gemserallv olemiionstra-
chemical olemonstra(i(In of strvl suilfalase.
ble by this metlsod! otslv ins tlse niuscosa of the Science 116: 539-543, 1952.
dusoslensusm also! uper j(junsunsi sum! in the kidney 7. RITENBERn, A. M., WotmAN, M., AND SEmnG-
MAN, A. M.: Comparative distnibumtioln of
cortex. Starvations resuslted ins decreased o’nszv-
succinsic dehydrogenase in six mammals and
msiatic stctivity in tlse itstestitsal niuscosa, vheretts nioldificatioms in the histolchemical technic.
J. Histochens. Cytochens. 1: 66-81, 1953.
imscreased aotivity was observes! after the inig(’s-
8. HLTENBIRG, A. M. AND SELnGMAN, A. M.: The
tiolni of food. hsistocbiemical demonsstration of acid 1)hOS-
llhatase by a post--incubation couspling tech-
BIBLI()( RAPHY msiqute. J. Histochem. (‘ytochens. 3: 455-470,
1. BAKER, J. It. AND FtSI-IMAN, W. H.: Effects (If 1955.
chlorstl-fornnal fixstt ions oIls preservatlols and 9. SEUGNnAx, A. M., TS0IU, K. C., RUTENBLRG,
localizations of five emizymes. .1. Histochens. S. H. ANn) COHEN, H. B.: Histochemical
(‘ytochem. 6: 85, 1958. demonsstrationi of l-nI-gluicuronidItse with a
2. COHEN, it. B., RUTENBIRG, S. H., Tsou, K. C., synt het-ic suslist rate. J. Histochem. (‘ytochem.
WOODBURY, M. A. ANLI SELIGMAN, A. M.: 2: 209-229, 1954.