THE FINE STRUCTURAL .LOCAUZATION . 1

AND BIOCHEMICAL ASSAY OF " .

NON’SPECIFIC ALKAUNE ANMQTD f I ~
PHGSPHATASES 1'" THE DEVELOP'NG *i’r'f: V1757

‘INTESTINE OF’TH‘E MOUSEFROMQ; .1;
BIRTH TD ADULTHOOD.’:J _ '

Thesis for the. Degree of Ph. D.
. MICHIGAN STATE UNIVERSITY, ,
GUSTAV ATTA OFOSU
1972

 

This is to certify that the

thesis entitled
The Fine Structural Localization and Biochemical
Assay of NOn-Specific Alkaline and Acid Phosphatases
in the Developing Intestine of the Mouse from Birth

to Adulthood presented by

Gus'nxv A-r-rA OFosu

has been accepted towards fulfillment
of the requirements for

Ph . D degree in Zoology

‘ W
:3 14" vaéi
Major professor

Date 7 )7 72/

0-7639

ABSTRACT

THE FINE STRUCTURAL LOCALIZATION AND BIOCHEMICAL
ASSAY OF NON-SPECIFIC ALKALINE AND ACID
PHOSPHATASES IN THE DEVELOPING
INTESTINE OF THE MOUSE FROM
BIRTH TO ADULTHOOD

BY

Gustav Atta Ofosu

Activity of alkaline phosphatase is found in
association with the brush borders of the intestine with
distributional changes occurring as the animal develops
from birth to adulthood. This is demonstrated by the use
of Naphthol AS-MX phosphate coupled with Hexazonium
pararosaniline. Post osmication for one hour results in
the formation of osmium black that appears electron dense
possibly at the site of enzyme activity. At birth the
apical portion of the microvilli are well stained with
little reaction at the basal portion. As the animal ages,
the degree of activity changes gradually going from the
apical to the basal regions of the microvilli with some
activity in the cytoplasm. By day 8 enzyme localization
can be demonstrated all along the microvilli reaching a

peak in intensity by day 15 to day 22. By day 30 there is

Gustav Atta Ofosu

observed a drop in the staining intensity which is
maintained through adulthood.

The spectrophotometric determinations for the
amount of inorganic phosphate liberated at an alkaline pH
indicates that there is minimal activity at birth with peak
activity occurring around the time of weaning from day 15
to day 22. After day 22, there is a steady decrease until
about day 30 and remains more or less the same throughout
adulthood.

Naphthol AS-TR phosphate served as the substrate
for demonstrating the acid phosphatase activity. Activity
is found to occur on the periphery of cytoplasmic vacuoles
or vesicles in the 1 day old animals as dense deposits.

By 8 days, the enzyme activity can be demonstrated within
the vesicles which tend to fuse and form bigger globules
reaching about 2.5 u in diameter by day 30. Assaying for
the amount of inorganic phosphate at an acid pH, it is
found that the lowest activity is at day l with a moderate
increase from day 8 to day 15. The peak activity occurs
around day 22 and drops markedly by day 30. Thereafter
the drop in the enzyme activity level does not go down to
the levels of the day 1 animals.

The overall level of acid phosphatase activity is
much less than that of alkaline phosphatase in the
intestine regardless of age. Histochemically, the

localization of the acid phosphatase and the alkaline

Gustav Atta Ofosu

phosphatase can be distinguished according to their

particular functional behavior.

THE FINE STRUCTURAL LOCALIZATION AND BIOCHEMICAL
ASSAY OF NON-SPECIFIC ALKALINE AND ACID
PHOSPHATASES IN THE DEVELOPING
INTESTINE OF THE MOUSE FROM

BIRTH TO ADULTHOOD

BY

Gustav Atta Ofosu

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology

1972

11 ACKNOWLEDGMENTS

I would like to express my deepest appreciation to
Dr. S. K. Aggarwal for his advice, academic help and
assistance in preparing this thesis.

Appreciation is also extended to Dr. J. R. Shaver,
Dr. R. N. Band, and Dr. R. Monroe for their assistance as
committee members.

I am grateful to Dr. R. Fennell for teaching me so
much about histochemistry and providing the experimental
basis for this research.

A special thank you is also extended to Miss Brenda
Alston, without whose invaluable aid and encouragement this
work could not have been accomplished. Brenda, please
taste my gratitude every hour of the day.

Finally, I would like to express my sincere thanks
to my wife and my children whose love, understanding, and

moral support helped to crown the success of my endeavors.

ii

TABLE

LIST OF TABLES . . . .
LIST OF FIGURES. . . .

LIST OF ABBREVIATIONS. .

INTRODUCTION. . . . .

General Background . .

Histochemical Techniques

OF CONTENTS

Acid Phosphatase Methods .

Alkaline Phosphatase Methods. . . . . . . .

Enzyme Chemistry. . .
MATERIALS AND METHODS. .

Tissue Preparation . .
Incubation Procedures .

Acid Phosphatase . .
Alkaline Phosphatase.

Electron Microscopy. .
Spectrophotometry . .

RESULTS . . . . . .
Light Microscopy. . .

Alkaline Phosphatase.
Acid Phosphatase . .

Electron Microscopy. .
DISCUSSION . . . . .

LIST OF REFERENCES. . .

Page

. . . . . . . iv
0 O O O O O O I V
. . . . . . . . ix
0 O O O O O O O 1
O O O O I I O O 1
I O O O O O O O 3
. . . . . . . . 5
7

O O O O O O O O 8
. . . . . . . . 12
O O O O O O O O 12
O O O O O O O 0 l3
. . . . . . . . 13
O O O O O O O 0 l4
0 O O O O O O O 15
O O O O O O O 0 l6
. . . . . . . . 21
O O O O O O O O 24
O O O O O O O O 24
O O O O O O I O 27
O O O 0 O O O O 31
O O O O O O O O 72
. . . . . . . . 81

iii

LIST OF TABLES

Table Page

1. Specific Experimental Data for Alkaline
Phosphatase Read in Percent Transmission
at 310 mu . . . . . . . . . . . . 68

2. The Average Percent Transmission Readings
for Alkaline Phosphatase and the Amount of
Liberated Inorganic Phosphate Based on
0.2 mg/ml Wet Weight of Tissue . . . . . 68

3. Specific Experimental Data for Acid
Phosphatase Read in Percent Transmission
at 310 mu . . . . . . . . . . . . 69

4. The Average Percent Transmission Readings for
Acid Phosphatase and the Amount of Inorganic
Phosphate Liberated Based on 0.2 mg/ml
Wet Weight of Tissue . . . . . . . . 69

iv

LIST OF FIGURES

Figure Page
1. Calibration Curve for Inorganic Phosphate . . 18
2. Light Micrograph of a Part of a Cross

Section From the Mouse Intestine Showing
the Villi and the Crypts . . . . . . . 23

3. A Low Power Electron Micrograph Showing Some
of the Epithelial Cells from a Villus. . . 23

4. A High Power Electron Micrograph of a
Representative Area Enclosed by the
Rectangle in Fig. 3. . . . . . . . . 26

5. An Electron Micrograph of a Section Through
the Brush Border of an Epithelial Cell
Showing the Microvilli Both in Longi-
tudinal and Transverse Section . . . . . 26

6. A Light Micrograph of a Glutaradeldehyde-
Fixed Frozen 10 u Thick Section of a 15
Day Old Mouse Duodenum Stained for
Alkaline Phosphatase With Napthol AS-MX/

Hexazonium Pararosanaline. . . . . . . 29
7. A Light Micrograph of a Section Adjacent to

that in Fig. 6 Serving as a Control . . . 29
8. Light Micrograph of a Section Adjacent to

that in Fig. 7 Showing Complete Inhibition
of Alkaline Phosphatase Activity After
0.05 M L-phenylalanine Treatment . . . . 29

9. Light Micrograph of a Glutaraldehyde Fixed
Frozen 10 u Thick Section of a 15 Day Old
Mouse Intestine Stained for Acid
Phosphatase With Napthol AS-TR/
Hexazonium Pararosaniline. . . . . . . 33

Figure Page

10. Light Micrograph of a Section Adjacent to
That in Fig. 9 Incubated in a Mixture
Without the Substrate . . . . . . . . 33

11. Light Micrograph of a Section Adjacent to
That Shown in Fig. 10 . . . . . . . . 33
12. Light Micrograph of a l u Thick Section

From a Tissue Block of a 22 Day Old

Mouse Duodenum Fixed in Glutaraldehyde,

Treated for the Demonstration of

Alkaline Phosphatase and Embedded in

Araldite . . . . . . . . . . . . 36

13. An Electron Micrograph of a Transverse
Section Through the Microvilli Showing
Alkaline Phosphatase Activity on and
Around the Plasma Membrane Only. . . . . 36

14. An Electron Micrograph of a Section From a
1 Day Old Mouse Duodenum Showing
Alkaline Phosphatase Activity at the
Apical Portion of the Microvilli Only. . . 38

15. An Electron Micrograph of a Control

Section Adjacent to the Section Shown

in Fig. 14. O O I O O O O O O 0 O 38
16. Electron Micrograph of a Section from an 8

Day Old Mouse Duodenum Showing Alkaline
Phosphtase Activity All Along the
MicrOVilli. O O O O O O O O O O O 40

17. An Electron Micrograph From a Section
Adjacent to the Preceding Section
Incubated Without the Substrate in the
Medium . . . . . . . . . . . . . 40

18, 19. Electron Micrographs of Sections from a 15
Day Old Mouse Duodenum Incubated in a
Mixture With and Without the Substrate
for Alkaline Phosphatase Respectively. . . 42

20, 21. Electron Micrographs of Sections from a 22
Day Old Mouse Duodenum Incubated in a
Medium With and Without the Substrate
for Alkaline Phosphatase Respectively. . . 44

vi

Figure

22, 23.

24.

25.

26.

27.

28.

29.

30, 31.

32.

Electron Micrographs of Sections from a 30
Day Old Mouse Duodenum Incubated in a
Medium With and Without the Substrate
for Alkaline Phosphatase Respectively
Showing Reaction Product on the Micro-
villi (Fig. 22) and no Activity in the
Control Section (Fig. 23) . . . . .

Electron Micrograph of a Section from a 60
Day Old Mouse Duodenum Incubated for the
Demonstration of Alkaline Phosphatase .

Electron Micrograph of a Section From a 60
Day Old Mouse Duodenum Incubated in a
Medium Without the Substrate . . . .

An Electron Micrograph of a Section From a
1 Day Old Mouse Duodenum Incubated With
0.05 M L-phenylalanine in the Substrate
Mixture for the Demonstration of
Alkaline Phosphatase. . . . . . .

An Electron Micrograph of a Section from a
60 Day Old Mouse Duodenum Treated the
Same Way as the Tissue in Fig. 26 . .

Electron Micrograph of a Section From a 1
Day Old Mouse Duodenum After Incubation
for Acid Phosphatase With Napthol AS-TR/
HPR O O O O O O O O O O O 0

Electron Micrograph of a Control Section
From a 1 Day Old Mouse Duodenum Incubated
in a Medium Without the Substrate for
Acid Phosphatase Products . . . . .

Electron Micrographs of Sections from an 8
Day Old Mouse Duodenum Incubated in a
Medium With and Without the Substrate
for Acid Phosphatase Respectively . .

Electron Micrograph of a Section From a 15
Day Old Mouse Intestine Showing Acid
Phosphatase Activity in Dense Bodies in
the Cytoplasm . . . . . . . . .

vii

Page

46

48

48

51

51

53

53

55

57

Figure Page

33. Electron Micrograph of a Control Section
From a 15 Day Old Mouse Intestine
Incubated in a Medium Without the
Substrate for Acid Phosphatase . . . . . 57

34, 35. Electron Micrographs of Sections From a 22
Day Old Mouse Duodenum Incubated in a
Medium With and Without the Substrate
for Acid Phosphatase Respectively . . . . 59

36. Electron Micrograph of a Section From a 30
Day Old Mouse Duodenum Showing Acid
Phosphatase Activity as Dense Lysosome-
Like Structures . . . . . . . . . . 61

37. Electron Micrograph of a Section From a 30
Day Old Mouse Duodenum Incubated in a
Medium Without the Substrate for Acid
Phosphatase . . . . . . . . . . . 61

38, 39. Electron Micrographs of Sections From a 60
Day Old Mouse Duodenum Incubated in a
Medium With and Without the Substrate
for Acid Phosphatase Respectively . . . . 63

40. An Electron Micrograph of a Section From a
1 Day Old Mouse Duodenum Incubated
With 0.01M Sodium Fluoride in the
Substrate Mixture for Acid Phosphatase . . 66

41. Electron Micrograph of a Section From a 60
Day Old Mouse Duodenum Treated the Same
Way as the Tissue in Fig. 40. . . . . . 66

42. Graph Representing the Amounts of Inorganic
Phosphate (Pi) in Each 0.2 mg/ml of Tissue
Homogenate Per Age Group for Both Acid
and Alkaline Phosphatase . . . . . . . 71

viii

LIST OF ABBREVIATIONS

C crypt
CM cell membrane
ER endoplasmic reticulum
LP lamina propria
M mitochondria
MG mucus secreting goblet cell
MV microvilli
N nucleus
TW terminal web
VAC vacuole
VES vesicle

ix

INTRODUCTION

General Background

 

Castro-intestinal tissue of vertebrates has long
been recognized as a rich source of many hydrolytic
enzymes such as acid and alkaline phosphatases,
dipeptidases, esterases, etc.

The small intestine is a complex organ divided into
3 segments: the duodenum, the jejunum, and the ileum. The
small intestine is composed of basic functional units
consisting of villi or finger-like projections and crypts,
located at the base of the villi and which contain
proliferative cells. After division, cells migrate out of
the crypt and onto the villi (Leblond and Stevens, 1948).
The villi are made up of highly differentiated cells and
are lined by a surface epithelium consisting of columnar
cells, which are the primary absorbing units of the small
intestine, and goblet cells that manufacture mucus and
secrete it into the intestinal lumen. The epithelial cells
sit on a basement membrane that separates them from the
connective tissue and the lamina propria. The lamina

propria forms the base of the villus and contains nodules

of lymphoid tissue, smooth muscle fibers, and the blood
and lymphatic vessels.

The study of the anatomical differences between
the crypts and villi (Mukherjee and Williams, 1967) has
enlarged our knowledge of the role played by each group in
the different functions of the intestine. A number of
enzymatic activities have been found predominantly in the
villi (VanGenderen and Engel, 1938; Dahlqvist and Nordstrom,
1966). The synthesis of DNA (Leblond and Messier, 1958) of
RNA (Shorter and Creamer, 1962) and of cholesterol
(Dietschy and Siperstein, 1965) has been reported to occur
in the crypts. Galjaard and Bootsma (1969) reported very
high esterase activity in the functional cells of the
villus and almost no activity present in the proliferating
cells. There was, however, a moderate esterase activity
in the non-dividing matured crypt cells. Thymidine kinase
was found in proliferating cells located in the crypts
while thymidylate phosphatase and adenylate deaminase were
distributed uniformly from the crypt to the villus
(Imondi, Balis, and Lipkin, 1969).

Lindberg and mean (1966) and Dahlqvist and Lindberg
(1966) studied the development of dipeptidase and alkaline
phosphatase activities in the rat small intestine and
correlated their findings with the maturation of the
mucosal cells. The association of alkaline phosphatase,

adenosine monophosphatase, and adenosine triphosphatase

with sites of absorption in the small intestine was
reported by Brown and Millington (1968). Moog (1962) and
Przelecka, Ejsmont, Sarzala, and Taracha (1962) have
related alkaline phosphatase to the absorption of nutrients
from the gut, and Barka (1962; 1963) has correlated acid

phosphatase to pinocytic and reverse pinocytic activities.

Histochemical Techniques

 

Contributions to the development and understanding
of the various techniques of enzyme histochemistry have
been numerous but those of Gomori (1941), Pearse (1954),
Defendi and Pearse (1955), Holt (1956), Nachlas, Young,
and Seligman (1957), and Burstone (1958) are especially
noteworthy.

Earlier histochemical methods for enzymes charac-
terized by a slight diffusion around the sites of enzymatic
activity have been replaced by more precise and discrete
localization. It has been recently recognized by Nachlas,
Prinn, and Seligman (1956) that most of the diffuseness
obtained in hydrolytic enzymes reaction was due to the
soluble and diffusible component of the enzyme (lyoenzyme).
Suitable methods of fixation and tissue preparation can
effectively allow this component to be diffused from the
tissue leaving behind for histochemical demonstration the
more precisely localized, less soluble component of the

enzyme (desmoenzyme). In 1955 it was demonstrated by

Sheldon, Zetterqvist, and Brandes that it is possible to
detect the reaction products of acid and alkaline
phosphatase activities at the ultrastructural level.

The functional and operational activity of a cell
is controlled by its component of catalytic proteins, the
enzymes. The sequence of events of an enzyme (E) substrate
(S) reaction to form a product (P) is usually given as

follows:

E + Sq——ES——9E + P

In enzyme histochemistry and cytochemistry, in order to
have the conditions necessary for the enzyme substrate
reaction to be observed in serial sections, the substrate
has to diffuse into the cell to form the primary product
(Pn). The product must then be captured by a coupling

agent (C) to form an insoluble color product (F).

Pn + c—'>PnC'—->F (colored)

The final reaction product must have electron scattering
properties to be seen with the electron microscope (Hunt,
1966).

The above rationale may have some obstacles in
demonstrating enzyme cytochemistry at the electron
microsc0pic level as discussed by Duijn, Pascoe, and
Ploeg (1967). It is the opinion of these authors that

both the rate of diffusion of substrate towards the site

of the enzyme molecule and the rate of final product
formation from the primary product influence the amount

of final product formed. According to Holt and O'Sullivan
(1953), if in fact the rate of final product formation

from the primary product is slow, this problem can be over-
come by increasing the concentration of the coupling
reagent to speed up the reaction. This would allow little
or no diffusion of the primary product. When the enzyme
reaction is followed as a function of time, the rate of
reaction can be expressed as the rate of disappearance

of the substrate and the rate of appearance of the product.

Acid Phosphatase Methods

 

A most well known procedure for localization of
acid phosphatase at the light microscopic level is the
lead procedure by Gomori (1941). The technique involves
forming an insoluble phosphate precipiate at an acid pH.
Although Gomori's method has been generally criticized
because of its limitations due to artifact formation, it
has provided a tool for studying the enzyme at the ultra-
structural level (Wachtel, e£_gl., 1959; Scarpelli and
Kanczak, 1965).

The azo dye technique for acid phosphatase which
was first introduced by Seligman and Manheimer (1949) did
not produce satisfactory results. The problems encountered

involved both diffusion artifacts and non-precision

binding. In 1952, Grogg and Pearse improved the technique
by using cold formalin fixed sections instead of acetone
fixed paraffin sections to cut down on diffusion artifacts.

Burstone (1958) compared the use of different
naphthol AS phosphates as substrates to demonstrate
phosphatases. He indicated that the AS-naphthols released
by enzymatic hydrolysis coupled over a wide range of pH
values as opposed to simple naphthols which coupled well
in the alkaline range.

According to Barka and Anderson (1962), the limi-
tations of using diazonium salts is due to the capture
reaction being too slow to prevent product diffusion.
However, by using freshly diazotized pararosaniline as a
coupler (Davis and Ornstein, 1959), a more insoluble
product could be formed. Barka and Anderson (1962) used
pararosaniline together with a-naphthol phosphate to
produce an amorphous azo dye insoluble in both lipids and
most inorganic solvents. The degree of coupling seemed
to be determined by different pHs, yielding color products
depending on whether the medium was acid, neutral, or
alkaline. These workers then combined the improved
methods of Burstone (1958) and the improved methods of
Davis and Ornstein (1959) to give artifact free acid
phosphatase localization. In 1964, Barka used this method
on the ultrastructural level and found that the combination

of pararosaniline and naphthol AS-TR was insoluble enough

to resist dehydration and embedding making it suitable to

be used in electron microscopic studies.

Alkaline Phosphatase Methods

 

In 1939 Gomori and Takamatsu independently intro-
duced the metal precipitation technique for alkaline
phosphatase localization. The technique involves the
release of phosphate ions by the action of alkaline
phosphatase on glycerophosphate to combine with calcium
ions to form an insoluble phosphate precipitate. The
precipitate is then converted to cobalt phosphate and then
to cobalt sulfide which indicates the sites of alkaline
phosphatase activity.

Menten, Junge, and Green (1944) introduced an azo
dye technique for alkaline phosphatase using Ca-B-naphthol
phosphate as substrate and diazotized a-naphthylamine as
the coupling agent to produce a red azo product with the
liberated naphthol. Several modifications were introduced
by Loveless and Danielli (1949), Manheimer and Seligman
(1949), and Gomori (1951). All modifications of the early
technique involve either stabilizing the salt to reduce
background staining, finding a more suitable substrate, or
getting optimal conditions in regard to temperature and
other conditions.

Burstone (1958) introduced the use of substituted
naphthol AS phosphates in simultaneous coupling techniques

and similar work was reported by Rutenburg, et al. (1958).

Modifications on Burstone's (1958) method by using
diazonium salt as a coupler have been tried (Hunt, 1966).

A technique employing metals as a capturing agent
was used by Mizutani and Barrnett (1965). They showed that
cadmium gave very good localization on the brush borders
of the kidney at an alkaline pH. In 1966, Hugon and
Borgers obtained excellent localization at the ultra-
structural level of alkaline phosphatase on the brush
borders of microvilli, the Golgi complex, and multi—

vesicular bodies by a direct lead method.

Enzyme Chemistry

 

Many workers have employed biochemical techniques
in conjunction with the various localization techniques
of histochemistry to present a more complete picture of
enzyme activity. In yitgg biochemical assays have long
been recognized as being advantageous to enzyme cytology.
Fortin-Magana, g£_al. (1970) used biochemical analysis to
study intestinal enzymes as indicators of cell proliferation
in diseased and healthy conditions. One of the methods
used is the technique of tissue fractionation and subse-
quent identification of enzymes in the fraction. Sloat
and Allen (1969) did a comprehensive study of two forms of
the enzyme acid phosphatase. These researchers wished to
localize the bound form and the soluble form of the
enzyme, both of which are associated with the lysosomal

fraction of regenerating rat liver. This was accomplished

by differential centrifigation of homogenates to obtain
the lysosomal fraction and using acrylamide gel electro-
phoresis and photometric assays on the fraction. Ide and
Fishman (1969) employed both techniques of tissue
fractionation and electron microscopy to study the
localization of some hydrolases in lysosomes and in the
endoplasmic reticulum. These findings showed that acid
phosphatase and B-glucuronidase are both associated with
an extralysosomal site.

Shnitka and Seligman (1971) have recently suggested
that electron cytochemistry and tissue fractionation are
mutually complementary. The former preserves the normal
morphological organization and the latter determines the
total amount of enzyme present in subcellular fractions.

Various simpler biochemical techniques such as
colorimetric procedures using tissue homogenates have also
been used in substantiation of histochemical findings.
Lowry and his co-workers (1954) reported a microcolorimetric
procedure that was sensitive enough to measure enzymes
using as little as 10 y of certain brain tissue.
Nordstrom, et a1. (1967) used tissue homogenates to
ascertain the differential localization of alkaline
phosphatase as correlated with its anatomical distribution
in the small intestine. The histochemical findings have
shown the activity to be at the apical portion of the

villi as opposed to the basal portion of the crypts. The

10

colorimetric procedure confirm this result. In 1968,
Fennell used tissue homogenates and colorimetry to
determine the intensity of several enzymes in lymphatic
tissue of White Leghorns chicken to substantiate the
histochemical findings. In many of the studies of Moog
(1961, 1966) and her co-workers (1966, 1968), whole
tissue homogenates were used to learn more about the
alkaline phosphatase activity in the small intestine of
the mouse. Both colorimetric and electrophoretic methods
were used.

In 1925, Fiske and SubbaRow proposed a colorimetric
method to determine both total phosphorous and inorganic
phosphate in biological material. The procedure was to
form a phosphomolybdate complex which would produce a
colored compound upon addition of a reducing agent.
Berenblum and Chain (1938a) devised a more sensitive
modification of the earlier procedure. They found that
many reducing agents would give color in the absence of
phosphate. Other sources of error were substances which
altered the acidity of the medium, substances that would
form complexes which were difficult to reduce, and
substances that would interfere with the phosphomolybdic
acid reduction (1938b). Isobutanol was used in this
method to extract the soluble and reducible phosphomolybdic
acid by shaking the extract with the reducing agent,

stannous choloride after which a blue color was obtained.

11

In 1965, Dreisbach discovered that the reducing agent
could be eliminated altogether since the isobutanol-soluble
phosphomolybdate is strongly absorbed at 310 mu.

Numerous studies of the distribution and locali-
zation of acid and alkaline phosphatases in mammalian
small intestine have been done (Ogawa, gt_al., 1962;
Barka, 1964; Liu, e£_al,, 1963; Dempsey and Deane, 1946;
Chase, 1963) but a comparative study of both histochemical
and biochemical features of acid and alkaline phosphatases
especially in developing mouse intestine has not been
reported. This study is undertaken, therefore, to provide
an integrating step to elucidate the relationship of the
biochemical activity during functional differentiation as
it relates to the ultrastructure of the small intestine
of the mouse during its development from birth to adult-

hood.

MATERIALS AND METHODS

Tissue Preparation

Swiss albino male mice supplied by Spartan
Research, Haslett, Michigan were randomly selected at
days 1, 8, 15, 22, 30, and 60. The animals were sacri-
ficed by cervical dislocation or by decapitation. After
killing the animal, an incision was made along the mid-
ventral line through the peritoneal sac to expose the inner
organs. In each instance a few millimeters of the
intestine were removed from the duodenal portion.

Tissues used for histochemical studies were mounted
on a microtome chuck with OCT Tissue-Tee (Ames) and were
dipped in solid COZ-Acetone for quick freeze. Sections
were cut on a Slee cryostat from 8-10 u in thickness.
Tissues to be used for electron microscopic studies were
fixed in 3% glutaraldehyde buffered to pH7.2 using
cacodylate buffer. After fixation the tissues were washed
in buffered 7.5% sucrose (pH 7.2) and stored at 4°C until
needed. 50 u thick sections were utilized for incubation

in the substrate medium.

12

13

Incubation Procedure

Acid Phosphatase

 

For localization of acid phosphatase a slight
modification of the Naphthol AS phosphate-hexazonium
pararosaniline (HPR) method was used (Barka and Anderson,

1962). The working medium was made up as follows:

 

 

Compound A Compound B
1.0 m1 pararosanilin 5 m1 veronal acetate
solution buffer pH 5.6
(1 g pararosanilin
hydrochloride dis- 12 ml distilled H O

solved in 20 ml dis- 2
tilled water and 5 ml 1.0 ml substrate (dissolve
concentrated HCL. 10 mg napthol AS-TR
Warm gently and filter.) Phosphate in 1-0 ml
N,N-dimethy1formamide.)
0.8 m1 4% sodium nitrite

1.6 ml 4% sodium acetate

Each compound was mixed separately and kept at 4°C; B was
poured into A just before incubation of the tissue. The
pH was adjusted to 5.6 with 1 N NaOH. The final volume
was brought to 25 ml with distilled water.

The frozen sections for light microscopy were
incubated in the above medium at room temperature for 30
minutes. After incubation, the sections were washed twice
in 2x distilled water, counterstained at a 1% solution of
methylene blue for approximately 2 minutes, washed, and
dehydrated in a graduated series of alcohols. The slides

were mounted in permount.

14

For electron microscopic observation, 50 u thick

sections were incubated at 4°C for 60 minutes.

Alkaline Phosphatase

 

For localization of alkaline phosphatase again, a
modification of Burstone's (1958) method was used for the
light microscopic studies (Hunt, 1966). The incubation

medium was prepared as follows:

 

 

Compound A Compound B
2 m1 pararosanilin 15 ml veronal acetate

solution (1 gm. buffer pH 9.2
pararosanilin
hydrochloride dis- 1.0 m1 substrate (dissolve
solved in 20 ml dis- 10 mg napthol AS-MX
tilled water and phosphate in 1.0 ml
5 ml concentrated HCl. N,N-dimethylformamide.)
Warm gently and
filter.)

0.8 ml 4% sodium nitrite

1.6 ml 4% sodium acctate

Mix Compound A and Compound B and pH adjusted to 9.2.
Final volume, brought to 25 ml with distilled water.
Incubation was for 25 minutes at room temperature. The
tissues were rinsed in distilled water followed by a
rinsing in 1% ammonium sulfonate, counterstained in 1%
methylene blue for 1 minute, washed, and dehydrated in a
graduated series of alcohols. The tissues were then air
dried and mounted in glycerine jelly.

For electron microscopic observation, 50 u sections

were inducated in the same medium as for the sections

15

used for light microscopy except the time of incubation was
changed to 30 minutes at 4°C.

Controls procedure for acid and alkaline phosphatase
involved the treatment of sections in the appropriate
mixtures but without the substrates. The second set of
controls involved the use of various inhibitors in the
medium. A 0.05 solution of L-phenylalanine was used for
alkaline phosphatase. For acid phosphatase, a 0.01M
solution of sodium fluoride was added to the standard
medium. The use of eserine disrupted the tissue structures

beyond the point of recognition.

Electron Microscopy

 

Incubated tissues were washed in cacodylate buffer
pH 7.2 with 7.5% sucrose and post-fixed in 1% or 2% osmium
tetroxide (buffered in cacodylate buffer pH 7.2) for 1 hour
or in vapors of osmium for 1/2 hour at 37°C. Embedding
was carried out in Araldite (Luft, 1961). Ultrathin
sections about 500 A were cut using an LKB ultratome III.
The sections were collected on 100 mesh copper grids and
examined using an Hitachi HU II E electron microscope
operated at 75 KV. Pictures were taken at desirable

magnifications.

16

Spectrophotometpy

 

In order to determine the total amount of inorganic
phosphate liberated in the enzyme-substrate reaction, the
procedure of Dreisbach (1965) was followed. The following

mixture was made in small test tubes:

1.5 ml 1 N H2804

0.2 ml 8% W/V ammonium molybdate

1.0 ml xylene-isobutanol (65:35 V/V)

0.25 ml unknown or standard containing 0.1 ug to

10 pg P as orthophosphate

Each tube was hand shaken for 10 seconds. The upper layer
was removed and read at 310 mu on a Beckman DU 2 Spectro-
photometer. The standard curve was prepared with known
concentrations of phosphate and the percent transmission
P0

was read. 2.193 9 KB in 500 ml distilled water gave

2 4
1 mg/ml of phosphate (Berenblum and Chain, 1938a).
Dilutions were made based on this value to give final
concentrations of 10 ug/ml, 1 ug/ml, and 0.1 ug/ml.
Logarithmic graph paper was used to construct the curve

5 and the

with the X-axis representing Pi in mg/ml x 10—
Y-axis the percent transmission (Fig. 1). The unknown
samples were read from the standard curve using linear
graph paper with the parameters being the age group on the
X-axis and the amount of liberated Pi in mg/ml x 10-5 on

the Y-axis.

Fig.

1.

17

Calibration curve for inorganic phosphate.

Known quantities (10 ug/ml, l ug/ml, and 0.1 ug/ml)
of inorganic phosphate (Pi) were read for percent
transmission (%T) at 310 mu wavelength using
Beckman D02 Spectrophotometer. The % T of unknown
samples and the amount of inorganic phosphate was

read directly and plotted in the graph (see
Fig. 42).

 

 

 

%T

18

"j

 

10L
0.5 1 5 10 50100 1000

mg/ml x10.5 (Pi) liberated

19

The sample for alkaline phosphatase assay was
prepared according to the method of Moog (1966). The
intestine was removed as previously described and placed
in a pre-weighed homogenizer. The wet weight was determined
as a Sartorius balance and the tissue was homogenized in a
minimal amount of distilled water. After homogenization
the concentration was brought to 10 mg/ml and transferred
into a small test tube. The mixture was kept in an ice
bath. 0.1 m1 of the sample was then diluted with 5 m1 of
distilled water in another small volume test tube, hand

shaken, and capped with Parafilm. This brought the working

concentration to 0.2 mg/ml. 0.5 m1 of dilute sample was
added to 1.0 m1 of B-glycerophosphate, 1.25 ml of
carbonate-bicarbonate buffer pH 9.6 and 0.25 ml of MgClz.
This preparation was slightly modified for acid phosphatase
samples. The addition of 0.5 m1 of dilute sample to

1.50 ml of tris-maleate buffer pH 5.2 and 1 ml of B-

glycerophosphate made up the working sample.

The blanks were compensated for the absence of
sample by the addition of an equivalent amount of buffer.
The controls were substrate free, with enough buffer being
added to make up the 3.0 ml volume.

Determinations were made on 5 aliquots of the
sample from each age group. The final results for both

standards and unknowns were averaged. All individual

20

tests for alkaline and acid phosphatases were done in such
a way that the reaction time for each sample was the same.
The incubation times for alkaline and acid phosphatases
were 45 and 30 minutes respectively.

All the light micrographs were prepared on a Zeiss
photoscope using Kodak Plus X film. The films were

developed in Microdol X.

RESULTS

The villi of the small intestine are finger-like
structures projecting into the lumen of the small in-
testine (Bloom and Fawcell, 1968). The epithelium lining
each villus is highly undulated and is of simple columnar
type. Two types of cells can be easily recognized in the
epithelial lining; the goblet cells and the columnar
absorptive cells. The goblet cells secret mucus into the
intestinal lumen, and the columnar absorptive cells are
the primary absoring units of the small intestine. The
core of each villus is formed by the lamina propria
(Fig. 2), a peculiar type of connective tissue that
resembles reticular connective tissue in that it contains
a stroma of argyrophilic fibers. Embedded in the fibers
of the argyrophilic stroma are large numbers of lymphocytes,
plasma cells, granular leukocytes, blood vessels,
lymphatic nodules, nerves and smooth muscle fibers. The
lining epithelium between the bases of the villi is
continuous forming the crypts of Lieberkuhn. The free
surface of the columnar epithelium is specialized to form

the striated border. The lateral surfaces of the columnar

21

Fig.

Fig.

2.

3.

22

Light micrograph of a part of a cross section from
the mouse intestine showing the villi and the
crypts. Representative area enclosed by the
rectangle is further enlarged in the next figure.
C, crypt; LP, lamina propria. (Frozen section
stained with 1% methylene blue) x 960.

A low power electron micrograph showing some of
the epithelial cells from a villus. Note that
the brushborder is formed by microvilli (MV).

The epithelial cells have an undulating cell
membrane (CM). A representative area of the
brushborder enclosed by the rectangle is shown at
a higher magnification in the next picture. N,
nucleus; VES, vesicles, x 8000.

23

 

24

epithelial cells are attached to each other by means of
plasma membranes in the form of interdigitating folds of
adjacent cell surfaces or appear as specialized adhesion
areas called tight junctions, terminal bars and desmosones.
The cytoplasm between the nucleus and the plasma membrane
contains a meshwork of fine filamentous materials and a
variety of cell organelles, such as, Golgi complex,
endoplasmic reticulum and mitochondria (Fig. 3). The
striated or brush border of the columnar epithelium is
revealed to be made up of countless closely packed finger-
like projections or microvilli. Each microvillus is
limited by the extension of the plasma membrane without
any detectable pore or discontinuities. The interiors of
microvilli show fibrilar strands which extend from each
microvillus into the terminal web (Fig. 4). This terminal
web is resolved with the electron microscope at high
magnification as a feltwork of exceedingly fine filaments
found beneath the striated border (Fig. 5). The outer
surface of the microvilli membranes is fuzzy or covered by
an extraneous coat believed to be some mucopolysaccharide

(Bloom and Fawcett, 1968).

Light Microscopy

 

Alkaline Phosphatase.--Using Naphthol AS-MX

 

phosphate/Hexazonium pararosaniline (HPR) as the incubation

medium, pH 9.2 for the demonstration of alkaline

Fig.

Fig.

4.

5.

25

A high power electron micrograph of a representative
area enclosed by the rectangle in Fig. 3. Note the
fibrilar strands (arrow) in each microvillus (MV)
extending into the terminal web (TW). CM, un-
dulating cell membranes of two adjacent cells; M,
mitrochondrion. x 30,000.

An electron micrograph of a section through the
brush border of an epithelial cell showing the
microvilli both in longitudinal and transverse
section. Note the fuzziness on the outer surface
of the microvilli due to a protein-polysaccharide
coat in the form of filaments (arrows) and the
terminal web (TW). x 37,500.

26

       

. . 6
“Vans 0".” 3&1":
I, as QmQ 05.4.2251

27

phosphatase on 8-10 u thick frozen sections, the reaction
product appears in the form of intense red coloration,
localized mainly on the brush border of the intestinal
epithelial cells (Fig. 6). Naphthol AS-MX phosphate is
split by the action of the enzyme into Naphthol AS-MX
(Equation 1) which is being captured by Hexazonium
pararosaniline (Equation 2).

The staining intensity does not seem to change
with age from one day through adulthood. The red color-
ation is fairly stable and resists dehydration through
acetone or alcohol series.‘ Upon osmication the Naphthol
AS-MX/HPR forms an osmium black product that is electron
dense. The intensity of the reaction product is con-
siderably reduced and appears anywhere from pale to brown
in coloration. The intensity may further be reduced after
dehydration in ethanol since osmium black is somewhat
water soluble. Sections incubated without Naphthol
AS-MX phosphate do not show any coloration product
(Fig. 7). The reaction product can also be blocked if
the sections are pretreated with 0.05 M L-phenylalanine a
specific inhibitor of alkaline phosphatase (Fig. 8).
Results are the same if 0.05 M L-phenylalanine is added

to the incubation medium.

Acid Phosphatase.--For the demonstration of acid
phosphatase Naphthol As-TR phosphate/HPR was used as the

incubation medium at pH 5.6. The Naphthol AS-TR phosphate

Fig.

Fig.

6.

8.

28

A light micrograph Fig. 7.
of a glutaraldehyde-
fixed frozen 10 u
thick section of a
15 day old mouse
duodenum stained for
alkaline phosphatase
with Naphthol AS-MX/
Hexazonium pararo-
sanaline. Note the
intense staining
only on the brush
borders of the villi
shown in cross
sections and longi-
tudinal sections.

x 250.

A light micrograph
of a section
adjacent to that in
Fig. 6 serving as a
control. This
section was treated
in a medium without
the substrate. To
bring out the tissue
details the section
was further stained
with 1% methylene
blue. x 450.

Light micrograph of a section adjacent to that in
Fig. 7 showing complete inhibition of alkaline
phosphatase activity after 0.05 M L-phenylalanine

treatment. x 450.

 

29

 

30

l|||||||||||||||CONNaZ O@O3

(Naphthol AS- MX phosphate)3

Alkaline Phosphatase

”1% CONHQ CH3

(Naphthol AS-MX)

Equation 1

 

N
111
NH2 N
+ HCl + NaNO2 > C
NH NH N N
2 2 111 111
N N
(Pararosanilin) (Hexazotized Form)

Equation 2

31

is split by the enzyme into Naphthol AS-TR (Equation 3)
which in turn is captured by Hexazonium pararosaniline to
give a red coloration. Napthol AS-TR/HPR upon osmication

forms osmium black .

\\\ OPO3Na2

/ CONH

Cl

CH3
Naphthol AS-TR

Equation 3

Acid phosphatase is present on the brush borders of the
epithelial cells. However, the intensity of staining

reaction is very low (Fig. 9). The true specificity of
the reaction is clearly indicated by the absence of any
reaction product if Naphthol AS-TR/phosphate is omitted
from the incubation medium (Fig. 10) and also after pre-
treatment of the section with 0.01 M Sodium fluoride--

a Specific inhibitor for acid phosphatase (Fig. 11).

flectron Microscopy

Preliminary survey of l u thick sections from
tissue blocks prepared for electron microscopic studies
indicated alkaline phosphatase activity mostly associated

With the microvilli (Fig. 12).

32

Fig. 9. Light micrograph of Fig. 10. Light micrograph of

a glutaraldehyde a section adjacent
fixed frozen 10 u to that in Fig. 9
thick section of a incubated in a

15 day old mouse mixture without the
intestine stained substrate. For

for acid phosphatase contrast in photo-
with naphthol AS-TR/ graphing, the
hexazonium pararo- section was stained
saniline. Note the with 1% methylene
staining confined to blue. x 450.

 

the brush borders of
the epithelial cells.
x 450.

 

Fig. 11. Light micrograph of a section adjacent to that
shown in Fig. 10. The section was pretreated
with 0.01 M Sodium fluoride to block the acid
phosphatase activity. x 450.

33

 

34

Examination of thin sections from the same blocks
in the electron microscope revealed enzyme activity on the
plasma membrane covering the microvilli while no enzyme
activity could be observed within the microvilli at any
stage during development (Figs. 13, l4, 16, 18, 20, 22,
24). This enzyme activity is demonstrated by the electron
dense osmium black, formed by a combination of osmium with
Naphthol AS-MX/HPR. In control sections of comparable
stages where Naphthol AS-MX/phosphate was omitted no
enzyme activity could be demonstrated (Figs. 15, l7, 19,
21, 23, 25).

At day l dense deposits of osmium black are
confined to the tips of the microvilli (Fig. 14). By day
8 one can observe enzyme activity in the form of dense
deposit of osmium black all along the borders of the
microvilli (Fig. 16). No enzyme activity could be observed
in the cytoplasm or associated with any cytoplasmic
organelles. From day 8 through day 22 there is observed
an increase in the amount of osmium black deposit around
the microvilli suggesting a probable increase in the
enzyme levels (Figs. 18, 20). By day 22 the first signs
of enzyme activity can be noticed in the cytoplasmic
vesicles (Fig. 20). These are more pronounced at day 30
(Fig. 22) and in the adult mouse (Fig. 24). Although with
the methods used it is not possible to ascertain the exact

amount of osmium black deposit on the microvilli, visual

35

Fig. 12. Light micrograph of a l u thick section from a
tissue block of a 22 day old mouse duodenum

fi fixed in glutaraldehyde, treated for the

E' demonstration of alkaline phosphatase and

" embedded in araldite. Note the enzyme activity

! only on the brush borders of the epithelial
cells. x 1000.

"fl‘i”

 

Aޤir

Fig. 13. An electron micrograph of a transverse section
through the microvilli showing alkaline

phosphatase activity on and around the plasma
membrane only. x 30,000.

 

36

 

37

Fig. 14. An electron micrograph of a section from a 1 day
old mouse duodenum showing alkaline phosphatase

activity at the apical portion of the microvilli
only. x 21,500.

 

 

Fig. 15. An electron micrograph of a control section
adjacent to the section shown in Fig. 14. The
tissue was treated the same way except that the
substrate was omitted from the incubation

medium. Note the absence of enzyme activity.
x 21,500.

 

39

Fig. 16. Electron micrograph of a section from an 8 day
old mouse duodenum showing alkaline phosphatase
activity all along the microvilli. x 15,000.

 

 

Fig. 17. An electron micrograph from a section adjacent
to the preceding section incubated without the
substrate in the medium. Note the alkaline

phosphatase activity is not demonstrated.
x 15,000.

40

 

41

m-fl

I“.
v "u I" . I‘-

 

 

Fig. 18, 19. Electron micrographs of sections from a 15
day old mouse duodenum incubated in a
mixture with and without the substrate for
alkaline phosphatase respectively. Note the
alkaline phosphatase activity confined to
the microvilli (Fig. 18) which is not
demonstrated if the substrate is omitted
(Fig. 19). x 15,000.

 

 

 

 

Figs.

20,

21.

43

Electron micrographs of sections from a 22
day old mouse duodenum incubated in a medium
with and without the substrate for alkaline
phosphatase respectively. Note the alkaline
phosphatase activity on the microvilli

(Fig. 20) which is not demonstrated in the
control section without the substrate in

the incubation medium (Fig. 21). x 15,000.

 

 

 

Tm,‘
A“

 

fl”.
3 1‘ ,-r 1-1-1"

Figs.

22,

23.

45

Electron micrographs of sections from a 30
day old mouse duodenum incubated in a medium
with and without the substrate for alkaline
phosphatase respectively, showing reaction
product on the microvilli (Fig. 22) and no
activity on the control section (Fig. 23).
Note the appearance of alkaline phosphatase
activity within the cytoplasmic vesicles
(arrow). x 15,000.

46

 

47

 

Fig. 24. Electron micrograph of a section from a 60 day
old mouse duodenum incubated for the demon-
istration of alkaline phosphatase. Note the
enzyme activity on the microvilli and in the
cytoplasmic vesicles (arrow). x 15,000.

 

 

Fig. 25. Electron micrograph of a section from a 60 day
old mouse duodenum incubated in a medium without
the substrate. Note the absence of any alkaline
phosphatase activity. x 15,000.

48

 

49

observations of the electron micrographs indicate a drop
in the amount of osmium black deposit per unit area from
day 22 onwards.

The specificity of reaction for the demonstration
of the enzyme, alkaline phosphatase, is further confirmed
by a pretreatment of the tissue with a specific inhibitor
such as 0.05 M L-phenylalanine (Figs. 26, 27).

Examination of thin sections from the tissue
blocks processed for the demonstration of acid phosphatase
reveal osmium black deposits associated with the membranes
enclosing the vacuoles or the dense bodies (Figs, 28, 30,
32, 34, 36, 38). Acid phosphatase could be demonstrated
at the brush borders after naphthol AS-TR/HPR (Fig. 9),
but could not be demonstrated after osmication (Fig. 28).
At day 1 the osmium black is mostly associated with the
membrane of the various vesicles with traces inside
(Fig. 28). By day 8 the various vesicles seem to fill up
with osmium black. As the animal ages the electron dense
bodies seem to increase in size (Figs. 32, 34, 36) possibly
by the fusion of smaller ones, since progressively fewer
smaller bodies per unit area are seen from day 22 through
the adult. The size of the electron dense bodies may
range from 0.1 u-2.5 p. The methods used are not useful
in quantitative determination of the number of such
bodies per cell which would require serial sectioning.

No such attempt was made to quantify the acid phosphatase

50

Fig. 26. An electron micrograph of a section from a 1 day
old mouse duodenum incubated with 0.05 M L-
phenylalanine in the substrate mixture for the
demonstration of alkaline phosphatase. Note the
total blockage of enzyme activity. The inhibitor
probably had a destructive action on the cellular
morphology at this age. x 21,000.

-11;

 

Fig. 27. An electron micrograph of a section from a 60
day old mouse duodenum treated the same way as
the tissue in Fig. 26. Note the tissue is able
to withstand the destructive action of the
inhibitor better. x 21,000.

 

51

  
      

‘ N .7 ~ :3 C
I -. . JV! ’.
t., I '7. "
"I. T 'r ..o '
. "I J y [I .
T \R I .
I " ‘ L i
y-. 1'

   

\ ‘i d

a r a» I i in
' x5“ ‘

 

52

Fig. 28. Electron micrograph of a section from a 1 day
old mouse duodenum after incubation for acid
phosphatase with Naphthol AS-TR/HPR. Note

"I dense deposits indicating the presence of acid
' phosphatase on the periphery of many cytoplasmic
vesicles or vacuoles. x 15,000.

 

Fig. 29. Electron micrograph of a control section from a
1 day old mouse duodenum incubated in a medium
without the substrate for acid phosphatase
products. Note the absence of any reaction.

x 15,000.

53

 

 

Figs.

30,

31.

54

Electron micrographs of sections from an 8
day old mouse duodenum incubated in a
medium with and without the substrate for
acid phosphatase respectively. Note the
dense granules of acid phosphatase activity
(Fig. 30) that are not seen if the substrate
is omitted (Fig. 31). x 15,000.

 

 

 

Fig.

Fig.

32.

33.

56

Electron micrograph of a section from a 15 day
old mouse duodenum showing acid phosphatase
activity in dense bodies in the cytoplasm.

x 15,000.

Electron micrograph of a control section from a
15 day old mouse duodenum incubated in a medium
without the substrate for acid phosphatase.
Note that the acid phosphatase activity is not
demonstrated. x 15,000.

 

 

 

 

Figs.

34,

35.

58

Electron micrographs of sections from a 22
day old mouse duodenum incubated in a medium
with and without the substrate for acid
phosphatase respectively. Note the acid
phosphatase activity in dense granules and
dense lysosome-like structures. (Fig. 34)
which is not demonstrated in the control
section without the substrate (Fig. 35).

x 15,000.

 

 

 

 

Fig.

Fig.

36.

37.

60

 

Electron micrograph of a section from a 30 day
old mouse duodenum showing acid phosphatase
activity as dense lysosome-like structures.

x 15,000.

Electron micrograph of a section from a 30 day
old mouse duodenum incubated in a medium without
the substrate for acid phosphatase. Note the
absence of acid phosphatase activity. x 15,000.

 

61

 

 

Figs.

38,

39.

62

Electron micrographs of sections from a 60
day old mouse duodenum incubated in a
medium with and without the substrate for
acid phosphatase respectively. Dense
bodies display acid phosphatase activity
(Fig. 38) which is not demonstrated in the
control section without the substrate
(Fig. 39). x 15,000.

 

63

 

64

positive granules. In the controls where Naphthol AS-TR
phosphate is eliminated from the incubation medium no
osmium black could be demonstrated (Figs. 29, 31, 33, 35,
37, 39).

The specificity of the reaction for acid phosphatase
is confirmed by the use of a specific inhibitor, sodium
fluoride, before or during incubation of the tissue

(Figs. 40, 41).

Spectrgphotometry

The isobutanol method for the extraction of in—
organic phosphate (Driesbach, 1965) based on 0.2 mg/ml
wet weight of tissue homogenate was used. The amount of
inorganic phosphate release is interpreted as a measure of
the acid or alkaline phosphatase activity, depending upon
the pH used. At a pH of 9.2 in 1 day old animals there is
very low alkaline phosphatase activity. At 8 days there is
a rise in activity. The peak in inorganic phosphate/
enzyme levels is reached by day 15. From 15 days to 22
days there is observed a slight drop from the peak. This
age represents the time of normal weaning from the milk
of the mother to solid food. After day 22 there is a
steady decrease until day 30. After this time, there seems
to be no further significant changes.

The spectrophotometric data are summarized in

Table 1. The amount of liberated inorganic phosphate as

 

 

Fig. 40.

Fig. 41.

65

An electron micrograph of a section from a 1 day
old mouse duodenum incubated with 0.1 M sodium
fluoride in the substrate mixture for acid

phosphatase. Note the total blocking of enzyme
activity. x 21,000.

Electron micrograph of a section from a 60 day
old mouse duodenum treated the same way as the
tissue in Fig. 40. Note that enzyme activity
can not be demonstrated. x 21,000.

L fl.__._—_—

66

‘ .. ”I, !

 

67

determined from the average percent transmission readings
for alkaline phosphatase are summarized in Table 2.

Assaying for the amount of inorganic phosphate at
an acid pH, of 5.2 it was found that the lowest activity
occurs at day 1 with an increase from 8 days to 22 days of
age. In contrast to alkaline phosphatase, the peak for
inorganic phosphate/acid phosphatase is found to be at 22
days of age. There is a marked decrease in the activity
by day 30. This level is maintained in the adult and
always stays at a higher level than found at 1 day.

Spectrophotometric data for acid phosphatase is
tabulated in Table 3. Average percent transmission
readings and corresponding levels of liberated inorganic
phosphate are summarized in Table 4.

The overall levels of acid phosphatase in all
cases observed is less than that of alkaline phosphatase
in the developing duodenum of the mouse regardless of the

age (Fig. 42).

68

TABLE 1

Specific Experimental Data for Alkaline
Phosphatase Read in Percent
Transmission at 310 mu

 

Day 1 Day 8 Day 15 Day 22 Day 30 Adult

 

 

Expt. 1 52 46 42 43 54 57
2 55 48 42 44 52 52
3 59 55 49 50 58 56
4 62 46 44 45 57 59
5 57 50 41 50 52 54
Average 57 49 43.6 46.4 54.6 55.6
TABLE 2

The Average Percent Transmission Readings for
Alkaline Phosphatase and the Amount of
Liberated Inorganic Phosphate Based
on 0.2 mg/ml Wet Weight of Tissue

 

Day 1 Day 8 Day 15 Day 22 Day 30 Adult

 

%T 57 49 44 46 55 56

mg/ml

x 10'5 79 200 450 325 87 84

 

Specific Experimental Data for Acid Phosphatase
Read in Percent Transmission

69

TABLE 3

 

 

 

 

 

at 310 mu
Day 1 Day 8 Day 15 Day 22 Day 30 Adult
Expt. 1 75 73 67 49 68 68
2 77 75 68 52 70 71
3 72 74 65 50 71 69
4 71 76 66 48 67 70
5 78 72 63 51 69 72
Average 74.6 74 65.8 50 69 70
TABLE 4
The Average Percent Transmission Readings for
Acid Phosphatase and the Amount of
Inorganic Phosphate Liberated
Based on 0.2 mg/ml Wet
Weight of Tissue
Day 1 Day 8 Day 15 Day 22 Day 30 Adult
%T 74 74 66 50 69 70
mg/ml
Pi
x 10-5 25 27 50 155 40 38

 

70

 

Fig. 42. Graph representing the amounts of inorganic
phosphate (Pi) in each 0.2 mg/ml of tissue
homogenate per age group for both acid and
alkaline phosphatase.

 

 

mg/ml x lO'5(Pi) liberated

450-

400-

350<

300-

2504

200'

1501

100.

50-

71

 

 

 

alkaline phosphatase

_ _ _- acid phosphatase

 

.01
O
.5
U

22

AGE (Days)

 

DISCUSSION

The results of this study suggest that many vari—
ables must be considered in studying the histochemical and
biochemical changes in the distribution of the enzymes,
alkaline and acid phosphatase during the development of
the small intestine of the mouse. This study is aimed at
examining the changes in the enzyme levels from birth to
adulthood and correlating these changes with nutritional
variations.

Some of the factors influencing the results of
enzyme localization at the electron microscopic level are
fixation, substrate, pH, temperature and the duration of
the incubation. Dempsey and Deane (1946) have indicated
that all of these factors will determine the type of
activity sites localized in any given region of the mouse
duodenum. Although their work was done on a light
microscope level, the concepts proved valid for reactions
at the electron microscopic level. Once light microscopy
established the presence of a reaction, more precise
methods were necessary for further study at the electron

microscope level. The method for artifact-free acid

72

73

phosphatase localization prOposed by Barka and Anderson,
1962 and Barka, 1964, using naphthol AS phosphate was
adopted. Fixation for all preparations was with
glutaraldehyde because it has been generally found to
preserve ultrastructure and enzymatic activity with
Optimal fidality (Sabatini, g£_al., 1963). Several pH
ranges for acid phosphatase localization were tried; pH
5.0-5.2, pH 5.4-5.6, and pH 6.0-6.2. Using naphthol
AS-TR/HPR at pH 5.6 proved to be very good. Promotion of
more efficient coupling probably occurs at this pH.
Barka and Anderson (1962) suggest pH 6.0-6.5 for efficient
coupling, but point out that about 54% of acid phosphatase
activity is inhibited at pH levels above 6.0. Variation
in temperature and duration of incubation seem to have
little effect. Incubation time at 37°C is shorter than
at 4°C. In the present studies the tissue preparation
for fixation, washing and storage were done at 40°C.
Transferring the tissue into a medium and incubating it
at 37°C distorted the tissue for electron microscopy. To
cut down tissue distortions as much as possible incubation
was carried on at 4°C for a longer period. Incubation
over 60 minutes caused a red precipitate in all media.
There is a minimal reaction observed for alkaline
phosphatase at birth as measured by biochemical assay.
Activity peaks around 15 days. Moog (1951) using a similar

biochemical assay interprets these quantitative changes

74

in the developing mouse intestine as a function of diet,
since until this age the animal is unable to digest
anything except milk. The rise in the enzymatic levels
probably represents a functional change in preparing for
solid food ingestion.

The above pattern is also shown histochemically as
indicated by this study. At 1 day of age the alkaline
phosphatase activity can be observed on the tips of the
microvilli. As the animal ages, the alkaline phosphatase
activity can be observed as dense dark bodies all along
the borders of the microvilli, and by day 30 the reaction
product is observed in the cytoplasm. Millington and
Brown (1967) in the case of rat intestine have shown that
there is no noticeable increase with age in the amount of
alkaline phosphatase present in the epithelial cells.
However there is a redistribution of enzyme from birth to
10 days post-weaning. They found a very strong reaction
around the brush borders and apical region of the cell.
As the animal aged, enzyme reaction product was found in
association with the lateral wall and the nucleus of the
epithelial cells. In the present studies there is no
evidence of reaction product in association with the
lateral walls or the nucleus. According to Chase (1963)
alkaline phosphatase activity has also been found in the
area of Golgi, on the brush borders and in the apical

cytoplasm of the epithelial cells of the mouse jejunum

75

but no mention was made of the animals' ages. Fredricsson
(1956), and Mizutani and Barrnett (1965), using rat small
intestine for the demonstration of alkaline phosphatase
also found discrete deposition of reaction product on the
membranes covering the microvilli. They also found some
enzyme activity in association with the Golgi complex.
In the present study no enzyme was associated with the
Golgi system any time during the present investigations.
It is possible that the quantity of the enzyme may be so
small that it is lost during the processing of the tissue
for electron microscopic observations. Such a possibility
was also suggested by Emmel as early as 1945.

Biochemical assays for acid phosphatase in the
present studies indicated a peak of activity at about 22
days, dropping significantly at 30 days after which the
level of activity is maintained through adulthood. The
functional significance of the strikingly high activity
of acid phosphatase in the duodenum at the peak of weaning
period and the decrease thereafter may be explained by the
progressive replacement of earlier cells by more mature
cells produced in the crypt after 21 days (Moog, 1951).
Millington and Brown (1967) using rat intestine from
birth to 10 days post-weaning for the demonstration of
acid phosphatase found two types of inclusions. One found
on the periphery of the vacuoles and vesicles and the

other associated with lysosomal structures. Similar

76

results are found in the present studies at day 1, where
the osmium black deposits are localized at the periphery
of vacuoles and vesicles, and at 8 days or after, when
lysosome-like structures can be observed. Transformation
of small vesicular bodies into large dense bodies by
fussion and condensation is generally accepted (Smith,
1969). Localization of acid phosphatase at different
sites during development, as an indication of two differ-
ent forms of the enzyme, is a matter of interpretation.
Acid phosphatase is known to be associated with the
lysosomes, a correlation in which many workers (Novikoff,
1961; Novikoff, 1964; Millington and Brown, 1967; and
Sloat and Allen, 1969) have used to identify lysosomes.
Nordstrom, g£_al. (1969), comparing acid phosphatase
activity in the small intestine of sackling and adult rats
found little or no change in the distribution profile.

A question to be considered in regard to both
enzymes, concerns the presence of multiple enzyme forms
or the same enzyme with functional differences. The
present histochemical methods available do not make any
distinctions between different forms of acid phosphatases
or alkaline phosphatases. Specific inhibitors used to
substantiate the validity of enzyme localizations also
make no distinctions among the various types of enzymes.
This is in agreement with Sloat and Allen (1969) who

observed that both soluble and membrane-bound forms of

77

acid phosphatase are inhibited by sodium flouride. As has
been pointed out, there is a difference in size and shape
of the dense bodies between day l and adult duodenal cells.
The heterogeneity of acid phosphatase activity may be
related to the dynamic changes which take place in the
lysosomal granules during their life cycle, or their
functioning may be correlated to the heterogeneity in the
acid phosphatase as released by various biochemical means
(Bresnick and Schwartz, 1968). The process of fussion

and degradation associated with the lysosomes may be more
pronounced in some cases within the same cell. This in
turn would result in a heterogeneity in the lysosomal
forms. Also the concentration of hydrolytic enzymes may
be greater in cases where lysosomes have not fused to form
a phagosome than in the ones that have. This may further
be accentuated by the size of the vacuole with which the
lysosome fuses. In view of the known nature of lysosomes
and the fact that acid phosphatase localization is used as
a marker for lysosomes, there are strong indications that
the characteristic acid phosphatase localization reported
in these studies is in fact associated with true lysosome.
However, it cannot be concluded that the dense deposits
seen at day l are exactly the same as the lysosome-like
structures seen later. According to Ide and Fishman (1969)
the question of specific versus non-specific acid
phosphatase has not been adequately answered. 8-

glycerophosphate as a substrate is supposedly specific

78

for lysosomal phosphatases, whereas phenylphosphate and
naphthol AS phosphates are supposed to be preferential
substrates by soluble fraction enzymes and microsomal
fraction enzymes, respectively. However these authors
state that there is much overlap in the biochemical and
cytological findings concerning specific versus non-
specific acid phosphatases and from this it cannot be
concluded that localization and activity solely depend on
the specificity of substrate. The localization technique
used for these studies was specific for acid phosphatase
and on the ultrastructural level revealed the sites of
activity, but was in no way designed to distinguish forms
of enzymes. The biochemical assay also indicated total
activity at an acid pH and not the forms of the enzyme.
Ide, ep_al. (1969) suggested that the properties
of an enzyme can be transformed in the process of digestion.
The present results indicate that the properties of the
enzyme, alkaline phosphatase depend upon the functional
state of the intestine as it develops and upon what is
being ingested. The demonstration of alkaline phosphatase
on the membrane of the microvilli suggests that its
function may be involved in the actual absorptive process
or the transport of hydrolysed substances across the apical
surface of the columnar epithelical cells. After weaning
another function may be intracellular metabolism and

transcellular transport, since cytoplasmic localization is

79

apparent at this time. Therefore in considering the
developmental process, there may be either different
enzymes or transformed states of the same enzyme. Moog
(1951) suggests that the biochemical differentiation of
the digestive organs can be directly correlated to the
functional changes necessary to adopt to the digestion of
solid food after a milk diet. If this reasoning is valid,
then the results of the present studies are in support of
this argument since after 15 days the eyes of the animal
are open, the mother begins to avoid the litter and the
food tray can be reached. However Moog (1961), Moog, g£_al.
(1966), and Moog, g£_§l. (1969) have shown that there are
two distinct forms of the enzyme in the duodenum of the
20 day old mouse demonstrated by the hydrolysis of phenyl-
phosphate in preference to B-glycerophosphate. They contend
that during infancy there exists only one form but
synthesis of a new form occurs at 15 days, which would
account for the increased level. The cytoplasmic in-
clusions seen in the present studies at 30 days in fact
may contain a different form of the enzyme. Nevertheless,
these studies can neither support nor refute the presence
of two distinct forms, since only total activity was
measured.

The role of hydrolytic enzymes and their functional
properties is a broad and complex field of study. Many

variables due to the properties of the enzymes themselves

80

create problems that must be considered in making accurate
judgments as to how the enzyme does what it does. However,
much information can be gained from results obtained from
constantly improved techniques for the localization of

enzymes at the ultrastructural level.

LIST OF REFERENCES

LIST OF REFERENCES

Barka, T. (1962) Cellular localization of acid phosphatase
activity. J. Histochem. Cytochem. 10: 231-232.

Barka, T. (1963) Fat absorption and acid phosphatase
activity in the intestinal epithelium of mice.
JAMA 183: 761-764.

Barka, T. (1964) Electron Histochemical localization of
acid phosphatase activity in the small intestine
of mouse. J. Histochem. Cytochem. 12: 229-238.

Barka, T., and Anderson, P. J. (1962) Histochemical
methods for acid phosphatase using hexazonium
pararosaniline as coupler. J. Histochem. Cytochem.
10: 741-753.

Berenblum, I., and Chain, E. (1938a) Studies on the
colorimetric determination of phosphate. Biochem.
J. 32: 286-294.

. (1938a) An improved method for the colorimetric
determination of phosphate. Biochem. J. 32:
295-298.

 

Bloom, W., and Fawcett, D. W. (1968) A text book of
Histology. W. B. Saunders Comp. Philadelphia.

Bresnick, E., and Schwartz, A. (1968) Functional dynamics
of the cell. Academic Press Inc., N.Y. '

Brown, A., and Millington, P. (1968) Electron microscope
studies of phosphatase in the small intestine of
Rana temporaria during larvel development and
metamorphosis. Histochemie 12: 83-94.

 

Burstone, M. S. (1958) Histochemical comparison of
naphthol AS phosphates for the demonstration of
phosphatases. J. Natl. Cancer Inst. 20: 601-615.

81

82

Chase, W. H. (1963) The demonstration of alkaline
phosphatase activity in frozen-dried mouse gut in

the electron microscope. J. Histochem. Cytochem.
11: 96-100.

Dalqvist, A., and Lindberg, T. (1966) Foetal development
of the small intestinal disaccharidase and alkaline
phosphatase activities in the human. Biol. Neonat.
9: 24-32.

Dahlqvist, A., and Nordstrom, C. (1966) The distribution
of disaccharidase activities in the villi and
crypts of the small intestinal mucosa. Biochim.
Biophys. Acta 113: 624-626.

Davis, B. J., and Orstein, L. (1959) High resolution
enzyme localization with a new diazo reagent,
"Hexazonium Pararosaniline." J. Histochem.
Cytochem. 7: 297-298.

Defendi, V., and Pearse, A. G. E. (1955) Significance of
coupling rate in histochemical azo dye methods for
enzymes. J. Histochem. Cytochem. 3: 203-211.

Dempsey, E. W., and Deane, H. W. (1946) The cytological
localization, substrate specificity, and pH optima
of phosphatases in the duodenum of the mouse. J.
Cell Comp. Physiol. 27: 159-179.

Dietschy, J. M., and Siperstein, M. D. (1965) Cholesterol
synthesis by the gastrointestinal tract: locali-

zation and mechanisms of control. J. Clin. Invest.
44: 1311-1327.

Driesbach, R. H. (1965) Submicrogram determination of
inorganic phosphate in biological material. Anal.
Biochem. 10: 169-171.

Duijn, P. van, Pascoe, E., and Ploeg, M. vander. (1967)
Theoretical and experimental aspects of enzyme
determination in a cytochemical model system of
polyacrylamide films containing alkaline phosphatase.
J. Histochem. Cytochem. 15: 631-645.

Emmel, V. M. (1945) Alkaline phosphatase in the Golgi
zone of absorbing cells of the small intestine.
Anat. Rec. 91: 39-45.

Fennell, R. A. (1968) Some histochemical and biological
observations on the lymphatic tissues of highly
inbred White Leghorns. J. Morph. 125: 281-302.

83

Fiske, C. H., and SubbaRow, Y. (1925) The colorimetric
determination of phosphorus. J. Biol. Chem. 66:
375-400.

Fortin-Magana, R., Hurwitz, R., Herbst, J. J., and Kretchmer,
N. (1970) Intestinal enzymes: Indicators of
proliferation and differentiation in the jejunum.
Science 167: 1627-1628.

Fredricsson, B. (1956) Alkaline phosphatase in the Golgi
substance of intestinal epithelium. Exptl. Cell
Res. 10: 63-65.

Galjaard, H., and Bootsma, D. (1969) The regulation of
cell proliferation and differentiation in intestinal
epithelium. Exptl. Cell Res. 58: 79-92.

Gomori, G. (1939) Microtechnical demonstration of
phosphatase in tissue sections. Proc. Soc. Exp.
Biol. Med. 42: 23-26.

Gomori, G. (1941) Distribution of acid phosphatase in the
tissues under normal and under pathologic conditions.
Arch. Path. 41: 362-364.

Gomori, G. (1951) Alkaline phosphatase of cell nuclei.
J. Lab. and Clin. Med. 37: 526-531.

Grogg, E., and Pearse, A. G. E. (1952) A critical study
of the histochemical techniques for acid phosphatase
with a description of an azo dye method. J. Path.
Bact. 64: 627-636.

Holt, S. J. (1956) The value of fundamental studies of
staining reactions in enzyme histochemistry, with
reference to indoxyl methods for esterases. J.
Histochem. Cytochem. 4: 541-554.

Holt, S. J., and O'Sullivan, D. G. (1953) Studies in
enzyme cytochemistry. I. Principles of cytochemical

staining methods. Proc. Roy. Soc. Series B. 148:
465-468.

Hugon, J., and Borgers, M. (1966) Ultrastructural locali-
zation of alkaline and phosphatase activity in the
absorbing cells of the duodenum of mouse. J.
Histochem. Cytochem. 14: 629-640.

84

Hunt, R. D. (1966) "Microscopic Histochemical Methods for
the Demonstration of Enzymes" in Selected Histo-
chemical and Histopathological Methods, Thompsos,
S. W. C. C. Thomas, Springfield, Ill.

Ide, H., and Fishman, W. H. (1969) Dual localization of
B-glucoronidase and acid phosphatase in lysosomes
and in microsomes. II. Membrane associated
enzymes. Histochemie 20: 300-321.

Imondi, A. R., Balis, M. E., and Lipkin, M. (1969) Changes
in enzyme levels accompanying differentiation of
intestinal epithelial cells. Exptl. Cell Res.

58: 323-330.

Leblond, C. P., and Messier, B. (1958) Renewal of chief
cells and goblet cells in the small intestine as
shown by radioautography after injection of
Thymidine-H into mice. Anat. Rec. 132: 247-259.

Leblond, C. P., and Stevens, C. E. (1948) The constant
renewal of the intestinal epithelium in the albino
rat. Anat. Rec. 100: 357-378.

Lindberg, T., and mean, C. (1966) Intestinal dipeptidases
activity in the small intestine of the rat as
related to the development of the intestinal
mucosa. Acta Physiol. Scand. 68: 141-151.

Liu, H., and Baker, B. L. (1963) The influence of
techniques and hormones on the histochemical
demonstration of enzymes in intestinal epithelium.
J. Histochem. Cytochem. 11: 349-364.

Lowry, O. H., Roberts, N. R., Wu, M., Hixon, W. S., and
Crawford, E. J. (1954) Quantitative histo-
chemistry of the brain. II. Enzyme measurements.

Loveless, A., and Danielli, J. F. (1949) A dye phosphate
for the histochemical cytochemical demonstration
of alkaline phosphatase, with some observations of
the differential behavior of nuclear and extra-
nuclear enzymes. Quart. J. Micro. Sci. 90: 57-66.

Luft, J. H. (1961) Improvements in epoxy resin embedding
methods. J. Biophys. Biochem. Cytol. 9: 408-414.

Manheimer, L. H., and Seligman, A. M. (1949) Improvement
in the method for the histochemical demonstration
of alkaline phosphatase and its use in a study of
normal and neoplastic tissues. J. Natl. Cancer
Inst. 9: 181-199.

85

Menten, M. L., Junge, J., and Green, M. H. (1944) A
coupling histochemical azo dye test for alkaline
phosphatase in the kidney. J. Biol. Chem. 153:
471-477.

Millington, F., and Brown, A. C. (1967) Electron
microscope studies of the distribution of
phosphatases in rat intestinal epithelium from
birth to ten days after weaning. Histochemie 8:
109-121.

Mizutani, A., and Barrnett, R. (1965) Fine structural
demonstration of phosphatase activity at pH9.
Nature 206: 1001-1003.

Moog, F. (1951) The functional differentiation of the
small intestine: II. The differentiation of
alkaline phosphomonoesterase in the duodenum of
the mouse. J. Exptl. 2001. 188: 187-208.

Moog, F. (1961) The functional differentiation of the
small intestine VIII. Regional differences in the
alkaline phosphatases of the small intestine of
the mouse from birth to one year. Devel. Biol.

3: 153-174.

Moog, F. (1962) Developmental adaptations of alkaline
phosphatase in the small intestine. Fed. Proc.
21: 51-56.

Moog, F. (1966) The regulation of alkaline phosphatase
activity in the duodenum of the mouse from birth
to maturity. J. Exptl. 2001. 161: 353-368.

Moog, F., Etzler, M., and Grey, R. D. (1969) The differ-
entiation of alkaline phosphatase in the small
intestine. Ann. N.Y. Acad. Sci. 166: 447-465.

Moog, F., and Grey, R. D. (1968) Alkaline phosphatase
isozymes in the duodenum of the mouse: Attainment
of pattern spatial distribution in normal develop-
ment and under the influence of cortisone or
Actinomycin D. Devel. Biol. 18: 481-500.

Moog, F., Vire, H. R., and Grey, R. D. (1966) The multiple
forms of alkaline phosphatase in the small in-
testine of the young mouse. Biochim. Biophys.

Acta 113: 336-349.

Mukherjee, T. M., and Williams, A. W. (1967) A comparative
study of the ultrastructure of microvilli in the
epithelium of small and large intestine of mice.

J. Cell Biol. 34: 447-461.

86

Nachlas, M. M., Prinn, W., and Seligman, A. M. (1956)
Quantitative estimation of 1yo- and desmoenzymes
in tissue sections with and without fixation. J.
Biophys. Biochem. Cytol. 2: 487-502.

Nachlas, M. M., Young, A. C., and Seligman, A. M. (1957)
Problems of enzymatic localization by chemical
reaction applied to tissue sections, J. Histochem.
Cytochem. 5: 565-583.

Nordstrom, C., Dahlqvist, A., and Joseffson, L. (1967)
Quantitative determination of enzymes in different
parts of the villi and crypts of rat small in-
testine. Comparison of alkaline phosphatase,
disaccharidases, and dipeptidases. J. Histochem.
Cytochem. 15: 713-731.

Nordstrdm, C., Koldovsky, 0., and Dahlqvist, A. (1969)
Localization of -galactosidase and acid phosphatase
in the small intestinal wall. Comparison of
adult and suckling Rar. J. Histochem. Cytochem.
17: 341-347.

Novikoff, A. B. (1961) In The Cell. J. Brachet and A. E.
Mirskey, eds. II: 423-488. Academic Press, Inc.,
New York, N.Y.

Novikoff, A. B., Essner, E., and Quintana, N. (1964)
Golgi apparatus and lysosomes. Fed. Proc. 23:
1010-1022.

Ogawa, K., Masutani, K., and Shinonaga, Y. (1962)
Electron histochemical demonstration of acid
phosphatase in the normal rat rejunum. J. Histochem.
Cytochem. 10: 228-

Pearse, A. G. E. (1954) A20 dye methods in enzyme
histochemistry. Int. Rev. Cytol. 3: 329-358.

Przelecka, A., Ejsmont, G., Sarzala, M., and Taracha, M.
(1962) Alkaline phosphatase activity and synthesis
of intestinal phospholipids. J. Histochem.
Cytochem. 10: 596-600.

Rutenburg, A. M., Bartnett, R. J., Tsou, K. C., Monis, B.,
and Teague, R. (1958) Histochemical demonstration
of alkaline phosphatase activity by a simultaneous
coupling technic using naphthol AS phosphate. J.
Histochem. Cytochem. 6: 90-91.

87

Sabatini, D. D., Bensch, K. G., and Barrnett, R. J.
(1963) Cytochemistry and electron microsc0py.
The preservation of cellular ultrastructure and
enzymatic activity by aldehyde fixation. J. Cell
Biol. 17: l9-58.

Scarpelli, D. G., and Kanczak, N. M. (1965) Ultra-
structural cytochemistry: Principles, limitations,
and applications. Int. Rev. Exp. Pathol. 4:
55-126.

Seligman, A. M., and Manheimer, L. H. (1949) A new method
for the histochemical demonstration of acid
phosphatase. J. Natl. Cancer Inst. 9: 427-434.

Sheldon, H., Zetterqvist, W., and Brandes, D. (1955)
Histochemical reaction for electron microscope:
Acid phosphatase. Exp. Cell Res. 9: 592-596.

Shnitka, T. K., and Seligman, A. M. (1971) Ultrastructural
localization of enzymes. Ann. Rev. Biochem. 40:
375-396.

Shorter, R. G., and Creamer, B. (1962) Ribonucleic acid
and protein metabolism in the gut. Gut. 3:
118-128.

Sloat, B. F., and Allen, J. M. (1969) Soluble and
membrane-associated forms of acid phosphatase
associated with the lysosomal fraction of rat
liver. Ann. N.Y. Acad. Sci. 166: 574-601.

Smith, R. E. (1969) Phosphohydrolases in cell organelles;
electron microscopy. Ann. N.Y. Acad. Sci. 166:
525-564.

Takamatsu, H. (1939) Histological and biochemical
detectionof phosphatase in tumors. Trans. Soc.
Path. Japan 29: 492.

VanGenderen, H., and Engel, C. (1938) Onthe distribution
of some enzymes in the duodenum and the ileum of
the rat. Enzymologia 5: 71-80.

Wachtel, A., Lehner, G. M., Mautner, W., Davis, B. J., and
Ornstein, L. (1959) Formalin fixation for the
preservation of both intracellular ultrastructure
and enzymatic activity for electron microscope
studies. J. Histochem. Cytochem. 7: 297.