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THE {SQERZYMES {I}? SERUM ALKAUNfi
FHGSPHATASE ééxé CATS

Tfiests for fire Degree of M. S.

{*eiifiikfiéi‘i S’i‘fifl'i Ui‘éi‘f’ifigii‘fi’
John Vii. Kramer

3.968

  
   

LIBRARY f'

missus Michigan 3mm {1
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Universaty 4" .

This is to certify that the

thesis entitled

Secondary Classroom Teachers' Awareness,
Perception and Attitude Toward Reading
in the Content Areas

presented by

Janet Easton Antcliff Haque

has been accepted towards fulfillment
of the requirements for

Ph D . Education
degree In

 

 

Samuel ”BJP‘E'Sti-eis?’ III
Date August 4, 1976

0-7639

 

 

 

 

ABSTRACT
THE ISOENZYMES OF SERUM ALKALINE
PHOSPHATASE IN CATS

by John W. Kramer

Total serum alkaline phosphatase and serum zymograms of alkaline
phosphatase were obtained from normal mature cats and kittens and from
kittens with experimentally produced biliary obstruction. Mean total
serum alkaline phosphatase was 1.61 i;1.9* Sigma units for 12 normal
kittens and 0.82 i;0.678* Sigma units for 10 normal mature cats. Only
a beta-1 globulin alkaline phosphatase was located in serum zymograms
of normal mature cats and kittens.

There was no increase in total serum alkaline phosphatase in 2
kittens with ligated common bile ducts. Serum alkaline phosphatase
zymograms demonstrated beta-l globulin and alpha-3 globulin alkaline

phosphatases in both kittens within 14 days of ligation.

 

* One standard deviation

THE ISOENZYMES OF SERUM ALKALINE

PHOSPHATASE IN CATS

By

John W. Kramer

A THESIS

Submi t ted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1968

ACKNOWLEDGEMENTS'

The author gratefully acknowledges the assistance and encourage-
ment of his advisor and friend Dr. S. D. Sleight. He also wishes to
express his sincere appreciation to Drs. G. R. Carter and R. F. Langham
for serving as members of his committee.

This research was sponsored in part by Sigma Chemical Company of

St. Louis, Missouri.

TABLE OF CONTENTS

INTRODUCTION 0 O O O O O O O O O O I O O O O . I O O O O O O O 0

LITERATURE REVIEW 0 I O I O O O O O O O O O i O O O O I 0 O O I 0

Chemistry. . . . . . . . . .». . . .1. . . . . . . . .
Histochemistry . . .7. . . . . . . . . .-. . . .'. .
Clinical Pathology . . . . . . . . . . . . . . .
Source of .Serum Alkaline Phosphatase . . . . . . . . .
Isoenzymes . . . . . . . . . . . . . . . . .p. . . . .
Isoenzymes in Disease. . . . . . . . . a . . . a . .
Review of Studies Concerned with Alkaline Phosphatase
of Cats . . . . . . .1. . -. . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . .

Specimens. . . . . . . .'. . . . . . . . . . . . . . .
Hematology . . . . . . . . . . . . . . . . . . . . . .
Serum Chemistries. . . . . . . . . . . . . . . . .
Positioning Enzyme Activity. . . . . . . . . . . . . .
Biliary Stasis . . . . . . . . . . . . . . . . . . .

RESULTS 0 O I. O O O O O O O O O O O 0 O O O O O O I O O O O 0

DISCUSSION. 0 O O O O O I O O O O O O O O O I O O O O O O 0

Normal cats. 0 o o o o o o o o ’0 o o o o o o o 'o o o o
Biliary StfiSiS o o o o o o 'o o o o o o o o o o o o o

BIBLIOGRAPHY O O O O O O O O O O I O O O O O O O O O O O O O

VITA.

Page

N

\OOUI-DUON

10
12.
12
12
12
16
16
18
21

21
22

24

27

33

Table

LIST OF TABLES

Clinical tests for total serum alkaline phosphatase. .

Factors for the conversion of total serum alkaline
phosphatase units. . . . . . . . . . . . . . . . . .

Normal serum alkaline phosphatase activity in the cat.

Effectors of alkaline phosphatase. . . . . . . . . . .

Page

25

25

26

26

LIST OF FIGURES
Figure Page
1 Electropherogram of normal cat serum. Upper strip is a 19

zymogram of serum alkaline phosphatase (arrow: site of
enzyme activity) and lower portion is stained,for protein

INTRODUCTION

Enzymes with the same catalytic properties and found within the
same species are termed isoenzymes. Introduction of the term isoenzyme
is probably a reflection of our advances in enzyme characterization
and, as advancement continues, systematic names will probably be applied
where numbers are now used.

Isoenzymes have been introduced as a means of monitoring disease
conditions in both research and clinical medicine. Frequently changes
in permeability of cell membranes are demonstrable by changes in serum
enzyme activity before clinical signs are apparent or changes are
detectable by means of the light microscope.

Isoenzymes of serum alkaline phosphatase have been identified in
many species and are presently used in human medicine as a means of
increasing specificity of diagnosis in cases of osteogenic and hepatic
diseases.

Cats appear to be the only animals in which biliary obstruction
does not result in great elevations of total serum alkaline phosphatase
activity. The reason for this is unknown. The purposes of this study
were to:

1. Establish normal total serum alkaline phosphatase activity
of cats.

2. Characterize isoenzymes of serum alkaline phosphatase of cats
by means of electrophoresis.

3. Study the relationship of serum alkaline phosphatase to hepatic

biliary obstruction.

LITERATURE REVIEW

Chemistry

Alkaline phosphatase is a phosphoric monoester hydrolase of
animals which demonstrates little or no specificity as to the radical
from which it hydrolyzes the phosphate. Its systemic name is ortho-
phosphoric monoester phosphohydrolase and it is capable of catalyzing
transphosphorylation as well as dephosphorylation (Enzyme Nomencla-

ture, 1964). Chemical reactions catalyzed are as follows:

Dephosphorylation
O O
l I
R - O - P - PH + H20 ----- R - OH + H0 - P - OH
I l
OH OH
Transphosphorylation
O O
I I
R - O - P — OH + R - OH ----- R - OH + R - O - P - OH
I I
OH OH

As Pearse (1961) has pointed out, the term alkaline in this enzyme's
name refers to the Optimum pH at which it functions in 31532, not-ya
3139, .Because optimum pH depends on substrate and substrate concentra-
tion, it is not possible to define clearly the true optimum pH for this
enzyme until the substrates on which it acts ig;ylgg.are known.

What was formerly referred to simply as serum alkaline phosphatase
is in fact a group of enzymes with similar activity. This will be

documented as the review proceeds. The International Union of

3
Biochemists Committee on Enzymes defines isoenzymes as multiple enzyme
forms having the same activity in a single species (Webb, 1964). Thus,
it would appear that alkaline phosphatase can be demonstrated in more
than one form. However, Wilkinson (1962) stated that because of rela-
tive nonspecificity and numerous sources of this enzyme it would be
better to regard it as a family of enzymes until a clearer definition
can be made.

Early realization that serum alkaline phosphatase is of signifi-
cance in clinical medicine led to development and use of a number of
quantitative tests suitable for use in clinical laboratories (Table 1).
These clinical tests differ primarily in the substrate used and, be-
cause of this difference, it is difficult to compare values obtained
by one method to those obtained by another (Tietz g£_§1,, 1967). How-
ever, it does become necessary at times to make a comparison between

values because of the limited amount of work done in this field (Table 2).

Histochemistry

 

Gomori (1939) and Takamatsu (1939) independently develOped a tech-
nique for the demonstration of alkaline phosphatase in tissues. In
this technique a calcium salt of the freed phosphate is produced which,
and in turn, reacts with silver or cobalt salt at the site of enzyme
activity. The resulting silver or cobalt phosphate is then changed
to the free metallic form which can be visualized. Since then numerous
other substrates have been introduced for this same purpose (Pearse,
1961). Phenyl and naphthyl phosphates are now the more common sub-

strates in use.

4

Histochemical studies of alkaline phosphatase activity have been
carried out with tissues of the cat (Gomori, 1941; Kritzler and Beaubien,
1949; Smith and Freeman, 1954; Wachstein, 1955; Martin, 1951).

Gomori (1941) found alkaline phosphatase activity in cats' pre—
capillary and capillary blood vessels; also spleen, thymus, lymph nodes,
lingual epithelium, stomach, small intestine, colon, liver, pancreas,
bronchi, lung, urinary bladder, testis, epididymis, prostate, ovary,
uterus, brain, and adrenal. Gomori (1941), Kritzler and Beaubien (1949),
Smith and Freeman (1954) and Wachstein (1955) reported activity in the
glomeruli of the normal cat. These same authors did not find activity
in the glomeruli of the normal dog, human, monkey, rabbit, guinea pig,
rat, gopher, hog, and groundhog.

Smith and Freeman (1954) observed an increase in cytoplasmic lipids
of the renal tubules in the mature cat as contrasted to the kitten and
half-grown cat. They suggested that alkaline phosphatase activity in
these cells was in direct proportion to the lipid material. These same
workers and Wachstein (1955) reported activity in association with the
brush borders of proximal convoluted tubules. This may be an indica-
tion of a part played by the enzyme in the active transfer of molecules

across membranes (Pearse, 1961).

Clinical Pathology
Robison (1923) reported alkaline phosphatase in bone which led to
his theory concerning the calcification of bony tissue. Gutman (1959)
reviewed the work carried out by numerous authors who have aSsociated
high serum alkaline phOSphatase activity with disease conditions of

man. The earliest reports following Robisonis (1923) dealt with conditions

5
which were osteogenic. Later, Roberts (1930) reported raised levels
associated with obstructions of the biliary tract and, to a lesser
degree, in hepatocellular disease. Elevated levels of serum alkaline
phosphatase were of significant use in preclinical diagnosis of osteo-
genic conditions and early biliary obstruction, as increases in activity
occurred prior to a bilirubinemia. .However,.the need for a means of

early differentiation of these two primary_diseases remains.

Source of Serum Alkaline Phosphatase

Armstrong and Banting (1935) found detectable alkaline phosphatase
activity in many normal canine tissue homogenates. By removing the
spleen, kidney and intestine they were unable to demonstrate a decrease
in serum activity. They concluded that these organs were not the source
of an appreciable amount of normal serum activity. Roberts (1930)
established that bile has alkaline phosphatase activity and postulated
that this is the route of elimination in man. Armstrong and Banting
(1935) ligated the common bile duct of the dog and produced increases
in serum alkaline phosphatase activity. This rise could be reversed by
eliminating the ligature.

Cats were used for similar studies by Cantarow'g£._l. (1936),
Flood‘gthgl, (1937), Thannhauser gthgl, (1937), Dalgaard (1948), and
Carlsten.gtnglr (1961). These workers.were only able to produce slight
or moderate initial rises of serum enzyme activity in cats with ligated
common bile ducts. The resulting rise in most cases did not exceed
what was considered as normal. Thannhauser gtngl. (1937) and Flood.gt

El, (1937) ligated the ureters as well as the common bile duct, and

this resulted in an increased total serum alkaline phosphatase.

6

Thannhauser gtual. (1937) reported that when ascorbic acid was
added to serum from cats with ligated common bile ducts in concentration
of 5 mg./ml., total alkaline phosphatase activity of the serum was in-
creased to levels as high as those found in dogs under similar condi-
tions. They suggested that the reason for low levels of serum activity
in cats with ligated common bile ducts was not because of the absence
of the enzyme but rather absence of an activator, such as ascorbic acid.

Flood gtngl. (1937) examined urine as well as serum from 12-cats
with ligated common bile ducts and found 8 which had transitory increases
in urine alkaline phosphatase activity. Only 5 of the 8 exceeded the
preoperative normal level.

A number of authors have determined normal levels of total serum

alkaline phosphatase of cats (Table 3).

Isoenzymes

Bodansky (1937a) reported results of work dealing with the relation—
ship of bile salts to alkaline phosphatase activity of bone, kidney,
and intestine. He concluded that bile salts inhibit the enzyme acti-
vity in bone and kidney but not that in intestine. This was probably
the first recognition that alkaline phosphatase is not a single enzyme.
Thannhauser gt 31. (1937), Cloetens (1939) and Bodansky (1948) studied
the effect of different amino acids and organic and inorganic salts on
alkaline phosphatase obtained from different tissues (Table 4). The
substrate used was beta-glycerolphosphate.

It has become possible to separate alkaline phosphatase from other

tissue components in a number of ways.

7

On a basis of solubility in nonionic solvents such as ethanol or
butanol, the enzyme has been separated from the tissues of dog and
man (Norton, 1950; Schlamowitz, l954a,b; Schlamowitz and Bodansky,
1959; Ahmed and King, 1959; Moss, 1962; and Peacock.gt_al,, 1963).

Zone electrophoresis has been carried out on and in such media
as paper (Taleisnik g£_al,, 1953; Baker and Pellegrino, 1954), agar
gel (Haije and de Jong, 1963; Stevenson, 1961) and starch gel (Hunter
and Markert, 1957; Kowlessar 32.21,, 1959; Rosenberg, 1959; Estborn,
1959; Lawrence gt_al,, 1960; Dubbs gt;a1,, 1960; Boyer E£”2l-: 1961;
Kowlessar §£_§l3, 1961; Paul and Fottrell, 1961; Moss gtflgl., 1961;
Moss and King, 1962; Moss, 1962; Robinson and Pierce, 1964). Specimens
most commonly used were serums of man with activity reportedly located
in association with alpha-2 and beta-l globulins. Boyer (1961) reported
the location of 16 bands of activity in serums of man, all of which
were not present in any one specimen. Stevenson (1961) worked with
normal canine serums, as did Lawrence g£_§l, (1960), who, in addition,
examined the serums of man, monkey, rabbit, guinea pig, rat, and mouse.
Paul and Fottrell (1961) noted a similarity of zymogram patterns in
serums from mice, perch, guinea pig, rat, frog, and pigeon.

Separation has also been carried out with a chromatographic tech—
nique (Fahey 33 31., 1958) and by ultracentrifugation (Ahmed and King,
1959).

Once the enzyme was isolated it was characterized in a number of
ways. Schlamowitz (l954a,b) and Schlamowitz and Bodansky (1959) pro—
duced antiserums in rabbits against alkaline phosPhatase of canine
intestine, human bone, and human intestine. By the use of these anti-

serums they demonstrated that bone was the source of about 40 to 59%

8
of the normal human serum alkaline phosphatase activity.

Some of the work carried out previously on serum and tissue homogenates
was repeated on individual fractions. Ahmed gt 31. (1959) demonstrated
that ions of Mg and Co were activators and Zn was an inhibitor of alka-
line phosphatase activity of alpha-2 globulin of human placenta, bovine
kidney, and dog intestine. They reported that amino acids gave variable
results as effectors of alkaline phosphatase in these same tissues.

Robinson and Pierce (1964) established that neuraminidase slowed
3 out of 4 bands of alkaline phOSphatase activity detected in serums of
man by starch gel electrOphoresis. The band unaffected by neuraminidase
was inhibited by L-phenylalanine, which is known to inhibit inteStinal
alkaline phosphatase. They speculated that the differences in enzymes
may be in the prosthetic portion.

Moss (1960a,b), Moss gtugl. (1961), and Moss (1964) introduced
characterization of alkaline phosphatase isoenzymes by the spectrofluoro-
metric technique. This technique monitors the reaction of the enzyme
with the substrate. By introducing different effectors in the presence
of certain substrates, the Michaelis constant is determined under dif-
ferent conditions for each of the isoenzymes.

A genetic association of certain isoenzymes with blood groups has
also been demonstrated. Arfors §t_§l, (1963), Beckman (1964) and Bamford
.2£._l° (1965) have established that number and location of zones of
activity produced by zone electrophoresis of human serums were associa-
ted with blood groups. Gahne (1963) demonstrated similar patterns of
activity between monozygous human twins. Randel g£_al, (1964) and Randel
and Stormont (1964) reported an association between the blood groups of

sheep and serum isoenzymes of alkaline phosphatase.

Isoenzymes in Disease

 

As had been previously recognized, serum alkaline phosphatase acti-
vity increased in diseases associated with bone and extra- and intra-
hepatic biliary obstruction in man (Gutman, 1959). Keiding (1959) used
starch block electrophoresis to study serums of persons with bone and
hepatic disorders. He reported an association between hepatic disorders
and an increase in serum enzyme activity in the alpha-2 and alpha-l
globulin positions. In instances of bone disease increases of activity
were in beta—1 globulin position. Cooke and Zilva (1961) studied a
patient with an unnamed neOplasm with no bone metastasis. Serum alka-
line phosphatase activity was increased and the rise was associated with
the alpha-1 and alpha-2 globulin. In man, Chiandussi _£__l, (1962) and
Hodson §£_al, (1962) related hepatic disorders with an increase in serum
alkaline phosphatase activity associated with faster-moving beta lipo-
protein. In human bone diseases such as Paget's osteomalacia and para-
thyroid osteitis, increases in serum enzyme activity were associated
with slower-moving beta globulin (Hodson 2£Hfll-: 1962).

Using agar gel electrOphoresis, Haije and de Jong (1963) recorded
three zones of alkaline phosphatase activity in serums of man. They
located the increase in activity on the cathode side of alpha-2 globulin
in serums from patients with Paget's disease and osteoplastic secondary
bone tumors. In cases of secondary hepatic tumors, reversible hepatic
cellular damage, and malignant reticulosis, the increases in serum
enzyme activity were in alpha-2 and alpha-1 globulin.

Other Workers have reported results similar to those listed above

for serums of man (Latner and Hodson, 1962; Nordeloft Jensen, 1964;

10
and Newton, 1967). In summary they were:
a. alpha—2 and alpha—1 globulin -- diseases with hepato—
biliary obstruction
b. beta-1 globulin -— bone diseases
Pulvertaft and Luffman (1967) examined serums of persons who had
had partial gastrectomy. They found that the increase in serum alkaline

phosphatase level in these patients was probably not intestinal in origin.

Review of Studies Concerned with Alkaline Phosphatase of Cats

Since the work of Cantarow E£Hél° (1935), Flood'gtual. (1937), and
Thannhauser gt_al, (1937), little has been done to examine the cause of
the lack of a sharp increase of serum alkaline phosphatase activity in
cats with experimentally produced biliary stasis.

Thannhauser 23421. (1937) put forward the concept that apparent
increases in serum alkaline phosphatase activity following biliary stasis
may be due to the increase of an activator and not to an increase in
enzyme concentration. They found that the addition of ascorbic acid
to serums of cats with complete biliary stasis increased activity to a
level comparable to that of dogs with similar conditions.

Dalgaard (1948) ligated the common bile duct of 10 cats and examined
their serums for activity. He described results similar to the earlier
works but suggested that it is a mistake to regard the increase in
activity as negligible or slight in view of low normal activity. Both
he and Flood.gt.§1, (1937) had also examined the enzyme's activity in
urine and found some increases, although not in all cases of biliary
stasis. Flood g£_§1, (1937) suggested urine as a pathway of elimination;

however, Dalgaard (1948) nephrectomized cats which had ligated common

ll
bile ducts and no appreciable increases in serum enzyme activity occurred.
Dalgaard (1948) reported urea as an inhibitor of alkaline phosphatase
activity. The resulting accumulation of urea following the nephrectomy
may have suppressed enzyme activity. Dalgaard (1948) reported a dif-
ference in total serum alkaline phosphatase levels between young and
adult normal cats. Higher levels were reported in cats less than 11
months of age than in adults. Pregnant cats at term also had higher
levels.

Carlsten gt a1. (1961) found a moderate increase in enzyme activity
in lymph of the billiary lymphatics of cats with biliary obstruction.
They recorded a high level of activity in serum from the hepatic vein
in cats with biliary obstruction.

Gibson (1952) reported the death of a 6-year-old cat as the result

of cholelithiasis. The calculi consisted of calcium salts of bilirubin.

MATERIALS AND METHODS

Specimens

Serum and whole blood samples were collected from 4 male and 6
female mature cats and from 7 male and 5 female kittens. Male cats with
undescended testicles were regarded as kittens and their female litter
mates were also regarded as kittens. At the time of sampling, kittens
were estimated to be less than 4 months of age. One sample was obtained
from each animal. All animals had at least one month's acclimatization

prior to sampling.

Hematology

Blood samples were obtained by cardiac puncture from the kittens
and jugular puncture from cats. Serums were collected by centrifuga—
tion from clotted samples. Three milliliters of whole blood was col—
lected in vials containing 0.05 ml. of a 7.5% aqueous solution of the
potassium salt of ethylenediaminotetraacetic acid. Complete blood counts
(CBC) were done consisting of hemoglobin determination by the cyanmet—
hemoglobin method, total leukocyte count with hemocytometer, packed
cell volume percentage by the microhematocrit method, and differential

leukocyte counts. Each determination was done in duplicate.

Serum Chemistries
The biuret reaction (Kolmer, 1959) was used to determine total

serum protein.

12

13

Total serum alkaline phosphatase activity was determined by use of
a commercially prepared kit.* The test incorporated p-nitrophenyl
phosphate as a substrate. The substrate is colorless, but the product
is yellow in an alkaline solution and has an optimum absorption of 400 mu.
The unknown serum sample was incubated for 30 minutes in a buffered so-
lution of substrate. Following incubation the solution was diluted with
0.02N NaOH and the optical density was determined in a spectrophotometer
at 410 mp. The Optical density was compared with a standard curve pre-
pared by use Of a known concentration of p-nitroPhenol. The reason for
using the wave length of 410 mp and not the Optimum of 400 mp was to
compensate for presence of hemoglobin. Hemoglobin has less absorption
at 410 mp than at 400 mp and p-nitrOphenol absorption is not appreciably
different over this range (Sigma, 1963). A serum of known alkaline
phosphatase activity was run as a control with each series.

Serum alkaline phosphatase activity was expressed in terms of Sigma
units per m1. of serum. One Sigma unit will liberate l‘pM Of p-nitrophenol
per 60 min. at 37 C. and pH 10.5.

Serum.was stored at -20 C. after total serum protein and total serum

alkaline phosphatase determinations were carried out.

Electr0phoresig. The technique of Crawly and Eberhardt (1962) was used
with some modification. The medium was changed from ionagar to agarose.**

A 1% agarose gel was prepared in pH 8.6 Veronal buffer*** of 0.037 ionic

 

* Phosphatase, acid, alkaline, prostatic. Sigma Chemical Co.,
St. Louis, Mo.

** Agarose, Bausch and Lomb, Rochester, N.Y.

*** Half strength Buffer B—2, Spinco Division, Beckman Instru-
ments, Inc., Belmont, Calif.

14
strength. Five milliliters of the agarose gel was pipetted onto a 16-
cm.-long strip of 35-mm. unperforated plain photographic film leader*
and allowed to set. A trench 3 cm. x 0.5 cm. was cut into the gel midway
along.and across the strip.

Electrophoresis was carried out in.a spinco-Durrum cell** from
which the paper support stand and wick supports were removed. A pH
8.6 Veronal buffer*** of 0.075 ionic strength was the electrolyte. For
purposes Of maintaining uniformity,the maximum number of strips the cell
would hold (8) was always placed in it. With strips in place 0.1 ml.
of serum was.pipetted into the trench.

Electrophoresis was carried out for 60 minutes at 150 volts.
Albumin migrates approximately 5 cm. from the point of application under
these conditions. 1

Each sample was fractionated at least twice, but never in the same
cell at the same time.

Once electrophoresis was completed the strips were removed and cut
in half lengthwise. One-half Of the electrophoregram was used for pro-

tein staining and the other for a zymogram.

Staining procedure. Procion Brilliant Blue, MPRS# was used as a protein

stain because of its high specificity and reduced background staining

(Fazekas de St. Groth, 1962).

 

* DuPont P-4OB Cronar, E.l, DuPont de Nemours Co., Cleveland, Ohio.
** Spinco Division, Beckman Instruments, Inc., Belmont, Calif.

*** Buffer B-Z, Spinco Division, Beckman Instruments, Inc.,
Belmont, Calif.

# Colab Laboratories, Chicago Heights, Ill.

15

Solution:
Conc. HCl 20 m1.
Absolute methanol 980 ml.
Procion Brilliant

Blue M—RS 5 gm.
Strips were immersed in the staining solution for 5 to 15 minutes.
Four 15-minute rinses in absolute methanol followed. One final 5-minute
rinse in distilled water was carried out to insure complete removal of

buffer salts in the agarose. Following rinses, the strips were air-dried.

Zymogram. A simple modification of the standard histochemical staining
techniques (Pearse, 1961) was used for locating alkaline phosphatase
activity on the electropherogram. One-half Of the electr0pherogram
not stained for protein was immersed in the substrate solution for 2
hours at 37 C. The period may be reduced when serum containing more
than 3 Sigma units of activity is used. Following incubation the strips
were rinsed in distilled water and immersed in a freshly prepared aqueous
solution of the diazonium salt Fast Red Violet LB Sa1t* (5-benzamido-
4-chloro-o-toluidine).

Substrate solution:

Na naphthyl AS-MX phosphate 100 mg.

Magnesium sulfate 602 mg.
Tris (Gomori) buffer pH 8.3 360 ml.
Dionized water 640 ml.

After staining, the strips were rinsed in distilled water, fixed in 70%

methanol for 15 minutes and air-dried.

 

* Sigma Chemical Company, St. Louis, Mo.

16

PositioninglEnzyme Activity

Once dry, electr0pherograms stained for protein were scanned by
the use of a Spinco Analytrol* with the Scan-A—Tron* attachment. A
slit width of 0.5 mm. and a B5 balancing cam were used. The absorption
wave length for Procion Brilliant Blue is 602 mu (Fazekas de St. Groth,
1962); however, because of the large amount of serum used and, in turn,
the relatively great amount of stain present a 500 mu interference
filter was used to reduce sensitivity. The zymograms were also scanned
under these same conditions; however, in some instances staining was too
faint to record on the scanning device but was apparent to the eye. In
such instances calipers were used to measure the distance from the point
of application to the stained band. This distance was used to accurately
position bands of enzyme activity on the zymogram in relation to areas
of protein separation.

Standardization of the technique was carried out with human and

dog serums.

Biliary Stasis
Two kittens between 3 and 4 months of age were anesthetized with
sodium thiamylal.** By aseptic surgery a midline abdominal incision
was made and the common bile duct located at the head Of the pancreas.
The duct was doubly ligated approximately 2 cm. above its entrance into
the pancreas. The incision was closed and the cats made uneventful

recoveries. Two preoperative serum and blood samples were taken.

 

* Spinco Division, Beckman Instrument Co., Belmont, Calif.

** Surital, Parke, Davis & Co., Ann Arbor, Michigan.

17
Serum samples were taken every second or third day postoperatively.
Total serum alkaline phosphatase levels were determined on each sample.
After 7 postoperative days total serum bilirubin was also determined
(Kolmer.g£”al., 1959). The kittens were necrOpsied l4 and 16 days
postoperatively. Upon examination it was observed that the common

bile duct of both kittens had been completely ligated.

RESULTS

Total mean serum alkaline phOSphatase for 12 kittens was 1.61 i
1.9* Sigma units and 0.82 1:0.678* Sigma units for 10 cats. Higher
levels of activity occurred in kittens than in mature cats (P‘<I.05).**

Alkaline phosphatase activity was detected on 18 of 22 zymograms.
Samples in which no activity was detectable were from mature cats with
low total enzyme activity. Only one band of activity was located in
each sample. It was located near the peak of beta-1 globulin (Figure l).

ElectrOpherograms Of 2 samples with well demarcated bands of enzyme
activity were stained with Oil Red 0 for lipoprotein. The band of
enzyme activity was located within the band of beta lipoprotein.

In some samples the band of enzyme activity was narrow and intensely
stained. It appeared as a dense band in the beta globulin (Figure 1).
This narrow band of activity was found in 8 out of 12 kittens and 1 out
of 7 mature cats. The denser-band was more common (P <:.05)*** in

serums Of kittens than in serums of mature cats.

 

* One standard deviation.
** T = 2.71.

*** As determined by the Chi-square Test, Chi-square value
of 4.91.

18

l9

 

.A’
' ‘3
8' p’ ‘d:1 cC,
Globulm Mb.

Figure 1. Electropherogram of normal cat serum.
Upper strip is a zymogram of serum alkaline phosphatase
(arrow: site of enzyme activity) and lower portion is
stained for protein.

20

Zymograms from 2 normal and 1 abnormal human serums were also pro-
duced. In 2 of these, more than one band Of enzyme activity was detected
in the same positions as Haije and de Jong (1963) had reported when ion-
agar was used.

Total leukocyte counts were relatively high in most cats. This
was considered a physiological leukocytosis on the basis of the increase
being due primarily to a lymphocytosis.

The total serum alkaline phosphatase levels of 2 kittens with
ligated common bile ducts were unchanged. The range of activity was 1.0
to 2.0 units. Serum bilirubin values were 27.9 mg./100 m1. serum for
kitten 101 and 18.9 mg./100 ml. serum for kitten 102, 14 days post—
operatively. Zymograms of preoperative serums demonstrated a single
band of activity in beta-1 globulin. Fourteen days postoperatively 2
bands were demonstrated in both cats' serums. One band was beta-l globulin

and the other in alpha—3 globulin.

DISCUSSION

Normal Cats

The single band of serum alkaline phosphatase in normal mature
cats and kittens in beta-l—globulin suggests that one of the two iso-
enzymes located in normal human and dog serums is absent or at too low
a level to detect by the method used here.

Proteins are known to exist in states of equilibrium in which a
single protein may be capable of deconjugating or conjugating with
another protein or nonprotein substance. The existing state is depen-
dent on many factors, one of which is protein concentration.

ElectrOphoresis separates and concentrates proteins. This change
in concentration provides an opportunity for protein to shift its state
of equilibrium from one form to another. This change in form may result
in alteration of mobility so that at low concentrations it may be found
in one position and in higher concentrations another. Regardless of
change, enzyme activity may or may not be altered.

Because cat serum has a low level of alkaline phosphatase, it was
found necessary to use a larger sample size than that used by workers
with other species' serums (Haije and de Jong, 1963; Stevenson, 1961;
Yong, 1967). To compare the effect of the size of the serum sample,
zymograms of human and dog serums were prepared with both large and
small samples. No appreciable difference was detected. Greater sample
size, then, is probably not the reason for a single band of activity in
zymograms of normal cat serum.

21

22

The source of beta-l globulin serum alkaline phOSphatase of normal
cats is speculative. Other workers using similar methods have also
found activity in this same region in serums of normal children and
puppies (Haije and de Jong, 1963; Stevenson, 1961; and Yong, 1967).
They reported alkaline phosphatase from normal bone in beta-l globulin
and because of normally high bone growth in children and puppies sug-
gested that beta-1 serum alkaline phosphatase is of bone origin. Beta-
1 globulin serum alkaline phosphatase is seldom seen in adults except
in cases Of an active osteoblastic disease (note literature review).

Higher total serum alkaline phosphatase in kittens than in mature
cats and no change in mobility as a result of maturity leads me to
conclude that at least half of the enzyme activity found in beta-1
globulin of kittens has its origin in bone. While it is difficult to draw
conclusions across species lines the work of Haije and de Jong (1963),
Stevenson (1961), Yong (1967), and Paul and Tottrell (1961) demonstrated

many similarities of alkaline phosphatase mobility between species.

Biliary Stasis .

The reason is unclear for the appearance of a second band of enzyme
activity without an increase in total enzyme activity in serums of cats
with biliary stasis. However, as was noted at the beginning of this
discussion, protein mobility may be altered by conjugation or deconju-
gation with other substances. In this case the other substances may
be related to biliary obstruction. There is also the possibility that
there is an absolute increase in alpha-3 globulin enzyme and a loss of
some beta-1 globulin enzyme.

Alpha-3 globulin of cats corresponds in mobility to alpha-2 globulin

of humans and dogs. It is alpha-2 globulin serum alkaline phosphatase

23
which is reported by Haije and de Jong (1963) and Yong (1967) as being
increased in biliary Obstruction in man. It is my personal Observation
that this is also true in dogs.
If again conclusions may be drawn across species lines, there is
reason to believe that the alpha-3 globulin serum alkaline phosphatase
may arise from liver. However, why there is not an increase in total

serum enzyme activity is not clear.

 

SUMMARY

Total serum alkaline phOSphatase and serum zymograms of alkaline
phosphatase were obtained from normal mature cats and kittens and from
kittens with experimentally produced biliary Obstruction. Mean total
serum alkaline phosphatase was 1.61 i;1.9* Sigma units for 12 normal
kittens and 0.82 i;0.678* Sigma units for 10 normal mature cats. Only
a beta-1 globulin alkaline phosphatase was located in serum zymograms
of normal mature cats and kittens.

There was no increase hitotal serum alkaline phosphatase in 2
kittens with ligated common bile ducts. Serum alkaline phosphatase
zymograms demonstrated beta-l globulin and alpha-3 globulin alkaline

phosphatases in both kittens within 14 days of ligation.

 

* One standard deviation.

24

25

Table 1. Clinical tests for total serum alkaline phosphatase

‘:—'

 

 

 

 

L A‘

 

 

 

 

Originator . p . Substrate; ' . ‘ ‘Unit

Bodansky, O. (1937) beta glycerolphosphate 1 mg.P/100 m1. serum/
60'

King-Armstrong (1934) phenylphOsphate 1 mg. phenol/100 ml.
serum/30'

Bessey-Lowry-Brock p-nitrophenyl phosphate 1,uM p-nitrophenol/

(1946) 100 m1. serum/30'

Sigma (1952) p-nitrophenyl phosphate 1,MM p-nitrOphenol/

1 m1. serum/60'

 

Table 2. Factors for the conversion of total serum alkaline phospha-
tase units

 

 

fir

a. Bessey-Lowry-Brock units to King-Armstrong: multiply

the former.by 2.5 (Cornelius and Kaneko, 1963).

b. Bodansky units to Bessey—LowryeBrock units: multiply

the former by 1.8 (Gutman, 1959).

 

26

Table 3. Normal serum alkaline phosphatase activity in the cat

 

 

Author

No. of Specimens

Units

 

Flood gt_a_1_. (1937)

Cantarow g£_al, (1936)

Dalgaard (1948)

Bloom (1957)

Bekemeier (1962)

21
45
24

(adult)

10

0.6 - 5.0 Bodansky units/
100 ml.

0.95 - 3.84 Bodansky
units/100 ml.

4.1 i 1.7 King-Armstrong
units/100 ml.

0.0 - 7.1 (mean 3.35)
Bodansky units/100 ml.

3.4 (1.9 - 7.9) King-
Armstrong units/100 m1.

 

Table 4. Effectors of alkaline phosphatase

 

1

 

Tissue Inhibitor ActivatOrs
Intestine, brain, high levels - L-alanine, The same as those
kidney L-glutamate, L-lysin, which are inhibitors

L-histidine and glycine at high levels are
KCN activators at low
levels. Mg
Serum Zn, Cu Fe, Mn, Co, Ni, Mg

 

Thannhauser 25.31, (1937)

Cloetens (1937)
Bodansky (1948)

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VITA

The author was born on August 17, 1935, in Dearborn, Michigan.
Primary and secondary education was in the Dearborn Public School
District. In 1952 he attended the Henry Ford Trade School and, be-
cause Of its closing one year later, returned to Dearborn High School
from which he graduated in 1954. Between September 1954 and June 1960
he attended Michigan State University, where he earned the B.Sc. degree
in 1958 and the D.V.M. degree in 1960. During his stay at Michigan
State University he was employed as Resident Assistant in the dormi-
tory system and histotechnician in neuroanatomy and endocrine assay.
During two summer terms he was employed by Parke, Davis and Company to
work in vaccine quality control in primates.

Following completion of the D.V.M. degree he practiced veteri-
nary medicine in southern Michigan until December 1960, at which time
he entered the service of the New Zealand Department of Agriculture as
a veterinary Officer in Whangarei, New Zealand. In April 1964 he left
New Zealand to join Michigan State University's Nigerian Program at
the University of Nigeria, Nsukka, as an Advisor in Veterinary Clinical
Pathology. During his period with the Nigerian Program he toured
veterinary diagnostic facilities and diploma teaching programs for
U.S.A.I.D. and Michigan State University in the Sudan, Kenya, and

South Africa.

33

34
In July 1966 he returned to Michigan State University, East
Lansing, and joined the Department of Pathology as an Assistant

Instructor and began his work for the M.S. degree in pathology.

IES

 

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