THE STUDY OF ENZYMES IN TISSUE SECTIONS*!
G. GOMORI, M.D.
From the Department of Medicine, The University of Chicago
Generally speaking, the importance of the microtechnical demonstration of
enzymes is essentially the same as that of any other special staining procedure.
Both kinds of technic uncover certain structural details which would not be
visible in slides stained with simpler routine stains. However, there is this
fundamental difference: whereas regular staining methods only show various
morphologic structures in different shades, thereby facilitating their recognition,
technics that demonstrate enzymes are of the nature of true histochemical reactions, actually identifying and localizing individual chemical substances in the
tissues. And that is where their real importance lies. There are good reasons
to believe that at least some of these chemically very active enzymatic substances
must have a physiologic function. Unfortunately, our knowledge of the nature
of their role is very limited. One approach to the problem would be the collection
of data on the amount and distribution of the enzymes under various normal,
experimental, and pathologic conditions in the hope of obtaining clues. A
secondary importance of the microtechnical methods for enzymes is their use
in diagnosis. It is a well established fact that tissues have a definite and
typical pattern of distribution of enzymes. Under abnormal conditions the
pattern may change in a characteristic way. Also, certain pathologic cells or
tissues, morphologically indistinguishable from each other, may be differentiated
on the basis of their enzymatic behavior. Such examples will be cited under the
individual enzymes.
Up to a few years ago only four enzymes were capable of histochemical demonstration by routine laboratory procedures. Three of them, phenol oxidase,
peroxidase, and dopa oxidase are oxidative while the fourth one, urease, is hydrolytic The last being primarily a plant enzyme and occurring only in minimal
amounts in one animal tissue, the gastric mucosa, will not be discussed. Recently, three more hydrolytic enzymes have become accessible to microtechnical
demonstration.
The purpose of this paper is to give a short review of these special technics,
including the chemical principles involved, the methods proper, and their uses.
I. OXIDATIVE ENZYMES
A. Phenol oxidases. There are several enzymes, not too well differentiated
from each other, in this group, and there are a number of histochemical methods
described for their demonstration. They all oxidize phenols under specified
conditions. The oxidation products are either of a dark shade and visible as
* Read at the Refresher Course conducted by the American Society of Clinical Pathologists in Chicago, March 3, 1946. Received for publication, March 21, 1946.
f This work was done under a grant from The Douglas Smith Foundation for Medical
Research of The University of Chicago.
347
348
G. GOMORI
such (nadi reaction,14'17 Loele's naphthol method 13 ) and/or intensely basophilic
and can be stained with a variety of basic stains, such as Graham's stain. 9 I t
should be remarked that there are good reasons to doubt the true enzymatic
nature of these oxidations and the accuracy of localization (Dietrich,2 Hollande10),
but since some research workers still support the enzymatic theory, it was decided
to include them in this presentation.
Phenol oxidases can be demonstrated mainly in blood smears although fair
results may be obtained with frozen or celloidin sections. In fact, some of the
phenol oxidases are heat resistant and demonstrable even in paraffin sections.
The most important of the oxidase reactions is the so-called nadi reaction of
Winkler and Schultze which is described in most textbooks of microtechnic.
It brings out intensely blue granules in myeloid elements, both mature and
immature while lymphoid elements remain unstained. It may be very helpful
in the differentiation of leukemic cells.
B. Peroxidases. They oxidize certain compounds in the presence of hydrogen
peroxide. The most important application of peroxidase reactions is the
demonstration of hemoglobin, both in red cells and their precursors, and of
hemoglobin deposited in renal tubules in cases of hemoglobinuria. However, in
the latter case the pigment rapidly loses its enzymatic activity and often becomes
entirely negative within a few days.
Two tests are used: the old benzidine test of Lepehne11 and the so-called zincleuco test of Lison.12 The latter is so much superior to the former that it should
be used exclusively.
The technic is as follows. The optimal fixation is Slonimski's fluid16 (2 to 4 per cent
potassium ferricyanide dissolved in 5 to 10 per cent formalin-saline) but fair results may be
obtained after plain formalin, Orth's or Zenker's fluid. Fixatives containing acid or alcohol
should be avoided. Frozen sections are more suitable than embedded sections. The
results after paraffin embedding are usually, but not invariably, satisfactory. The stain'is
prepared by dissolving 0.5 to 1 per cent of either patent blue, acid violet or acid fuchsin,
in the given order of preference, in 2 per cent acetic acid. A few grams of zinc dust are
added to each 100 cc. of the solution which is then heated until the dye is decolorized to a
faint yellowish shade. This leuco-dye will keep in the ice box almost indefinitely and can be
regenerated in case of darkening by simply heating the mixture. For use, about 10 cc. is
filtered, and to the filtrate is added 0.5 cc. of acetic acid and 1 cc. of commercial (3 per cent)
hydrogen peroxide. The slides are flooded with this mixture for 2 or 3 minutes and then
rinsed. Hemoglobin stains selectively and intensely in the shade of the dye used. The
section is counterstained as desired, dehydrated and mounted for permanent preservation.
C. Dopa oxidase1 oxidizes dioxyphenylalanine to melanin. In pathology it is
of little significance The reaction may be used to distinguish true vitiligo from
postinflammatory leucoderma by the fact that in the former, the basal cells do
not contain the enzyme while in the latter, they do. Occasionally an amelan-.
otic melanoma may be recognized by the demonstration of dopa oxidase in its
cells. The technic itself can be found in textbooks of microtechnic.
I I . HYDHOLYTIC
ENZYMES
This group includes alkaline phsophatase, 3 • 4 -16 acid phosphatase 6 and
lipase. 7,8 The chemical principle of their demonstration lies in the resistance of
ENZYMES IN TISSUE
349
these enzymes to dehydration by acetone and subsequent embedding in paraffin.
The sections are incubated with a suitable este.r substrate. At the sites of
enzymatic activity the substrate will be hydrolyzed, with the production of free
acid. The ions of the acid are trapped in situ by the presence of certain metal
ions with which the acid combines to form an insoluble precipitate. This
precipitate is then transformed into a colored, easily visible compound. In the
case of the phosphatases, the substrate is glycerophosphate, buffered at the
indicated pH; in the case of lipase, a water soluble stearic or palmitic ester of
mannitan or polyglycol.
The optimal fixative for this group is chilled acetone. Most other fixatives
destroy these enzymes almost immediately. Therefore in routine study of this
group of enzymes, a bottle of acetone should be kept in the ice box, and thin
slices of tissue fixed in it as promptly as possible. However, excellent results are
often obtained with tissues fixed as late as 24 hours after death. Since paraffin
sections of acetone-fixed tissues have a tendency to break up into pieces when
floated on warm water they should be handled very carefully and only lukewarm
water should be used. One way of imparting resistance to the sections is by
impregnating the tissue blocks with 5 per cent acetylcellulose in acetone before
transferring them to benzene. The temperature of the paraffin oven must not
exceed 56°C, and the pieces must not be exposed to this temperature for longer
than 2 hours. This is especially important in the case of acid phosphatase which
is relatively sensitive to heat. Embedding can be hastened by the use of reduced
pressure. A piece of rubber tubing, connected through a safety bottle to a water
pump and introduced into the oven through one of its air holes, will serve as a
simple suction apparatus. The melted paraffin is kept in a wide-mouthed bottle
which has a tight-fitting stopper, with a piece of glass tubing in its single hole.
The end of the rubber tubing is attached to the glass. Average sized pieces of
tissue are easily embedded within one hour, using 3 changes of paraffin, each for
20 minutes. The first and the last changes are made in open dishes while the
second change is made in the vacuum bottle.
The technic is as follows. Up to the stage when the slides are incubated with the substrate the method is the same for all enzymes of this group.
1. Fix thin slices of tissue in chilled absolute acetone and keep them in the ice box from
12 to 24 hours. Dehydrate in 2 changes of absolute acetone, from 6 to 12 hours each, at
room temperature.
2. (Optional). Impregnate blocks for 24 hours in a 5 per cent solution of acetylcellulose
(Eastman's "low viscosity, high acetyl," No. 4644) in acetone.
3. Drain pieces rapidly. Transfer into 2 changes of benzene, i hour each.
4. Embed in paraffin.
5. Cut sections from 4 to 8 microns in thickness, float them on lukewarm (30 to 35 C.)
water and mount on slides.
6. Allow slides to dry. Melt paraffin by placing them in the oven for 10 minutes.
7. Run sections through xylene and alcohols to distilled water.
D. Alkaline phosphatase.
8. Incubate slides in the following mixture at 37 C. for 1 or 2 hours:
2 per cent solution of sodium barbital,
2 per cent solution of sodium glycerophosphate
25 cc. each;
350
G. GOMOBI
2 per cent solution of calcium chloride
5 cc.
2 per cent solution of magnesium sulfate
2 cc.
distilled water
50 cc.
9. Wash under the tap.
10. Immerse in a 1 or 2 per cent solution of any soluble cobalt salt (chloride, nitrate,
acetate, etc.) for 5 minutes.
11. Rinse thoroughly in several changes of distilled water.
12. Immerse in a dilute solution of yellow ammonium sulfide (a few drops to a Coplin
jarful of distilled water) from 1 to 2 minutes.
13. Wash. Counterstain as desired. Dehydrate and mount.
Sites of alkaline phosphatase activity stain black.
E. Acid phosphatase.
8. Incubate slides in the following mixture at 37 C. from 1 to 24 hours:
Molar acetate buffer pH 5 (100 cc. of 13.6 per cent CH3COONa-3H20 + 50 c c ; 6 per cent
acetic acid) 30 cc.; 5 per cent lead nitrate, 10 cc.; distilled water, 60 cc.; add slowly, while stirring, 2 per cent sodium glycerophosphate 30 cc.
Shake well, allow to stand for a few hours. Keep in the ice box. Before use, filter a
small amount and dilute with 2 or 3 parts of distilled water.
9. Rinse slides thoroughly first in distilled water, then in 2 per cent acetic acid, followed
again by distilled water.
10. Immerse in dilute ammonium sulfide as above.
11. Wash. Counterstain as desired. Dehydrate and mount.
Sites of acid phosphatase activity stain brown-black.
For some unknown reason, the staining for acid phosphatase sometimes turns out patchy,
occasionally even negative, when it should be positive. This seems to happen especially in
cases when the pieces have been exposed to the temperature of the paraffin oven for more
than an hour, or when the temperature of the oven is over 56°C.
F. Lipase.
Sections of tissues embedded without the use of acetylcellulose must be protected with
a thin layer of collodion after the last alcohol (blot slide; flood with a \ to 1 per cent solution of collodion in alcohol-ether; drain; dip in 95 per cent alcohol) because some lipase may
diffuse out of the tissue and give a blurred picture.
8. Incubate slides in the following mixture at 37°C. for 6 to 12 hours:
Stock solution I: Glycerol, 150 c c ; 10 per cent solution of calcium chloride, 50 cc.
M/2 maleate buffer pH 7 to 7.4 (5.8 Gm. of maleic acid dissolved in a mixture of 94 cc.
of 4 per cent sodium hydroxide and 6 cc. of water), 50 cc. distilled water, to make, 1000 cc.
Stock solution II:
5 per cent aqueous solution of Tween 40 or 60 (Atlas Powder Co., Wilmington, Del.) or
of Product 81 (Onyx Oil and Chemical Co., Jersey City, N. J.)
Both stock solutions, after addition of about 0.02 per cent of merthiolate, keep in the ice
box for many months.
For use, add 2 cc. of stock solution II to 50 cc. of stock solution I.
9. Rinse slides in distilled water.
10. Transfer to a 1 or 2 per cent solution of lead nitrate for 10 minutes.
11. Rinse in repeated changes in distilled water.
12. Immerse in dilute ammonium sulfide as above.
13. Wash; counterstain lightly with hematoxylin and eosin; dehydrate in alcohols; clear
in gasoline or tetrachloroethylene and mount in clarite dissolved in the same solvents. Do
not use toluene or xylene which will cause fading of the slides.
Sites of lipase activity are stained golden brown.
ENZYMES IN TISSUE
351
Any preformed insoluble calcium salt, present in the tissues, will stain very
much like the precipitate formed by enzymatic action. To avoid confusion,
these precipitates either have to be removed from the slide before incubation or
duplicate slides have to be stained with Kossa's silver stain and compared with
the incubated ones. Precipitates can be removed by treating the slides with a
citrate buffer of pH 4.5 to 5 for about 15 minutes. This method can be used
Only in the case of acid phosphatase or lipase; alkaline phosphatase, being acid
sensitive, will be very much weakened, if not destroyed altogether, by the treatment. It is also possible to stain, by a modification of the technique, 5 the preformed calcium salts and those produced by enzymatic action in different shades.
This latter modification is somewhat complicated and not recommended for
routine use.
Dark brownish pigments may also be mistaken for a positive enzyme reaction;
however the distinction is easy since pigments are visible in the nonincubated
slide whereas sites of enzymatic activity are not.
Some diagnostic applications of the stains for hydrolytic enzymes follow.
Alkaline phosphatase. Osteogenic sarcomas and Ewing's tumor are intensely
positive and can be distinguished from other tumors of similar morphology,
which are negative. The phosphatase picture of the nephritic kidney shows a
typical decrease in both intensity and extent from that of the normal kidney.
Although the number of cases observed so far is too small to permit definite
conclusions, there seems to be a relationship between the functional capacity and
the phosphatase picture of the kidney.
Acid phosphatase. Cancer of the prostate and its metastases can be easily
recognized by their extremely intense reaction. Whereas other tissues, even if
positive for this enzyme, require an incubation time of several hours for a good
positive reaction, prostatic epithelium, both normal and neoplastic, will usually
betray itself by an intense staining within one hour. Giant cell tumors of the
bone, while negative for alkaline phosphatase (except for the walls of small blood
vessels), are usually positive for the acid variety.
Lipase. Hepatomas are lipase-positive whereas cholangiomas and metastatic
tumors of the liver are negative. Squamous cell carcinomas are negative except
for some (not all) of those arising from the esophagus or the bronchi.
As mentioned in the introduction, the importance of the technics described
lies not in their limited diagnostic use but in the fact that they may open up a new
avenue of research in experimental pathology.
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G. GOMOEI
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