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ABSTRACT
ALKALINE PHOSPHATASE ISOENZYMES

IN NEONATAL JAUNDICE

BY

Joanne K. Gahan

Measurements of serum isoenzyme levels of alkaline phosphatase were.
made on 2 groups of jaundiced infants: (1) those with transient "physio-
logical" jaundice and (2) those with jaundice due to fetal—maternal
incompatibilities leading to hemolysis. Assuming that relatively few of
those with physiologic jaundice would manifest some degree of immaturity
of the hepatic excretory enzyme systems and that most of those with hemo-
lytic jaundice would show varying degrees of the same immaturity, the
experiment was designed to reveal any association of immature enzyme
systems, glucuronyl transferase and alkaline phosphatase in this instance.

The data revealed no associated decrease in the hepatic fraction of
alkaline phosphatase isoenzyme, but an unexpected decrease in the osseous

fraction of alkaline phosphatase was encountered in infants with hemolysis.

ALKALINE PHOSPHAIASE ISOENZYMES

IN NEONATAL JAUNDICE

By

Joanne K. Gahan

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1971

ACKNOWLEDGEMENTS

My sincere appreciation and gratitude is expressed to:

W. E. Maldonado, M.D., Director of Laboratories, Edward W. Sparrow
Hospital, for allowing me freedom in my work schedule to complete the
requirements for this degree; for his support and guidance throughout my
course of study, and for allowing me to use reagents and equipment.

A. E. Lewis, M.D., my advisor, for his continual counseling, guidance
and support in achieving this degree.

C. C. Merrill, D.V.M., Ph.D., Chairman, Department of-Pathology,f£or
the opportunity to enter this program and complete my research project
outside Michigan State University.

R. B. Fby, Ph.D., Technical Director of Laboratories, Edward W.
Sparrow Hospital, for his assistance in the selection of a research
project; his help in securing reagents; and for his time and support in
the completion of the research undertaken.

Drs. M. Jones, M.D., and A. F.-Kohrmen, M.D., my advisory committee.

F. K. Neumann, Administrator of Edward W. Sparrow Hospital, for the
financial support received during the graduate program.

My colleagues, for their invaluable advice, encouragement and support.

11

INTRODUCTION . .~. . . . .
Bilirdbin . . .
Phosphatases. .*. .

HISTORICAL REVIEW. . . . . .

MATERIALS AND METHODS. . . .

Sources of Specimens.

TABLE

Total Alkaline Phosphatase.

Me thOd O O O O

Standardization.

OF CONTENTS

Alkaline Phosphatase Isoenzymes

Reagents and Apparatus .

MéthOd o o o 0

Sample Application.

Detection .

Elution of Activity . .

Standard_Curve
Experiment. . . . . .

RESULTS. 0 o o o o o o o o 0

DISCUSSION 0.. o o o o o o 0'

SUMMARY AND CONCLUSIONS. . .

BIBLIOGRAPHY o o o o o o o 0'

APPENDIX 0 o o o o o o o o .-

iii

Page

12
12
12
12
12
13
13
13
13
14
15
15
16
21
27
30
31
34

35

Table

LIST OF TABLES

Electrophoretic analysis of alkaline phosphatase isoenzymes
in. normal" infants 0 O O O O O O O 0 O O O O O O O O O O O O

Electrophoretic analysis of alkaline phosphatase isoenzymes
in infants with an ABO incompatibility. . . . . . . . . . . .

Electrophoretic analysis of alkaline phosphatase isoenzymes
in infants with Rh incompatibilities. . . . . . . . . .-. . .

Tabulation of the mean and standard deviation (S. D. ) of
alkaline phosphatase fractions in "normal" and "abnormal"
neWboms. O O O O O C O O C I O O O O C O O O C Q C O O C O 0

Mean values obtained for "normal" and "abnormal" newborns

and the results of the application of the t-test for unpaired

data. 0 O ‘0 O O O O .0 I O O O O O O I. O O O I. O O O O O O O 0

iv

Page

21

23

24

25

26

LIST OF FIGURES
Figure Page

1 A diagram of the proposed site of action of the maternal
antibodies on the neonatal osteoblasts. . . . . . . . . . . . 28

 

INTRODUCTION

This thesis problem was designed to evaluate the relative degrees of
maturation of certain critical hepatic functions in the neonate.' All of
the data were collected on newborns showing varying degrees of hyperbili-
rubinemia. The principal comparisons in this study are limited to the
relationship of serum alkaline phosphatase isoenzymes and hyperbilirubinemia
in-2 groups of newborns. The groups were separated on the basis of immuno-
hematological evidence of fetal-maternal incompatibility leading to exces-
sive hemolysis. Those infants showing no incompatibility were designated
"normal" infants, displaying physiologic jaundice in varying degrees, while
the "abnormal" group displayed a defined fetal-maternal incompatibility
leading to hemolysis. The results obtained, in some instances, were
unexpected.

Cord bloods were tested electrophoretically for the presence of the
isoenzyme fractions of alkaline phosphatase. These data reveal a definite

change in total alkaline phosphatase levels in the 2 groups analyzed.

Bilirubin
The degree of hyperbilirubinemia present in the neonate is directly
related to the maturity of the newborn liver and the maturity of the enzyme
systems involved in the degradation of hemoglobin by-products.
Aged or damaged red blood cells are readily.phagocytosed by the
reticuloendothelial (RE) cells anywhere in the body, but particularly in.

the liver, spleen and bone marrow. The heme portion of the released

2
hemoglobin is converted in the RE cells, by a series of chemical changes,
to bilirubin. It was originally believed that bilirubin was carried in
the blood in the form of bilirubin-globin. However, it is now recognized
to be carried in the blood loosely bound, partly to albumin and partly to
al-globulin (Hoffman, 1970).

The bilirubin in normal plasma, although it may circulate in loose
combination with proteins, acts as free (indirect) bilirubin. This free
bilirubin is insoluble in water. When serum bilirubin passes through the
polygonal cells of the liver, it is converted, at least in part, to
glucuronide and, as such, is excreted into the bile. About 80% of the,
bilirubin conjugates with glucuronic acid to form'bilirubin glucuronide;
an additional 10% conjugates with sulfate to form bilirubin sulfate. The
final 10% conjugates with a variety of other solubilizing substances.

This converted or conjugated (direct) bilirubin is now highly soluble.

A small portion of the soluble bilirubin formed by the hepatic cells
returns to the plasma. This is then either re-excreted by the liver cells
directly into the liver sinusoids or it is reabsorbed into the blood from
the bile ducts. Regardless of the exact mechanisms by which bilirubin
reenters the blood, this results in the presence of a small portion of
soluble bilirubin in the body fluids.

Jaundice is a syndrome characterized by hyperbilirubinemia and yellow-
ish pigmentation of the sclerae, skin and mucous membranes. Occasionally,
the predominant color is green because of biliverdin, an intermediate
product of hemoglobin degradation. Jaundice is predominantly of 2 types:
(1) retention jaundice, in which there is overproduction of bilirubin.
caused by excessive hemolysis, the liver being unable to remove bilirubin
as fast as it is produced; and (2) regurgitation jaundice, in which bile

mechanically finds its way back into the blood stream because of either

3
intra-hepatic or post-hepatic obstruction of the bile ducts.

The mechanisms involved in the production of jaundice are not com-
pletely clarified. There is probably an overlapping of the 2 types of
jaundice in any individual case (Watson, 1937).

Jaundice, caused by a deficiency of the glucuronyl-transferase, is
a common occurrence in the neonate. The rate of formation of bilirubin
glucuronide in the polygonal cells of the liver determines the rate of
hepatic excretion of bilirubin. Although the fetal liver has a poorly
developed gucuronyl-transferase system, and the enzyme system gradually
improves during fetal development, it is frequently inadequate at the time
of birth. During fetal life, the plasma bilirubin is removed by the
placenta, apparently by a glucuronide forming mechanism (Claireau, 1960).
After birth, the enzyme system in the newborn liver develops rapidly and
becomes adequate in a week or two. In the immediate neonatal period,
however, all.infants have some hyperbilirubinemia, with approximately
30% having a level above 6 mgs./dl., producing visible jaundice in the
newborn. In the absence of any hemolytic factor, the jaundice usually
does not appear in the first 24 hours, is usually self-limiting and is
seldom associated with bilirubin levels above 10 mgs./dl.

However, physiologic neonatal hyperbilirubinemia may-not be entirely
due to inadequate glucuronyl-transferase. Some of it may be due to:'

(l) inhibiting effect of maternal pregnaediol present in the breast milk;
(2) increased enterohepatic recirculation of bilirubin because of the
absence of bacteria in the gastrointestinal tract which convert bilirubin-
to urobilinogen; and (3) formation of bilirubin from sources other than
hemoglobin (Brown, 1962). When, because of extraneous factors causing
excessive hemolysis, the amount of bilirubin formed is much greater than

normal, the serumrinsoluble bilirubin may rise above 20 mgs./dl. The

4

brain cells of the newborn infant may be sensitive to such levels of
unconjugated bilirubin, resulting in the condition known as "kernicterus."
This condition is characterized by cerebral manifestations with convulsions,

and is frequently fatal.

Phosphatases

The blood plasma of normal man contains representatives of 2 classes
of "nonspecific" phosphomonoesterases, one exhibiting optimal activity
at pH 9 with substrate in high concentration, and the other at pH 5,
hence alkaline and acid phosphatase. The proteins endowed with these
enzymatic properties are separated with the alpha-globulins by alcohol
fractionation of the plasma proteins (Edsal, 1951).

The "non-specific" phosphatases, unlike the "specific" phosphatases
(such as glucose-6-phosphatase) which are more selective in their sub-
strates, act upon the orthophosphoric monoesters of a.wide variety of
phenolic, alcoholic, carbohydrate and other compounds. Two types of
reactions are catalyzed. In one, the enzyme acts as the hydrolase, with
cleavage of the P-O bond and removal of the phosphoryl group to liberate
inorganic orthophosphate (Axelrod, 1956):

3 2 3 4
The second type of reaction, a transphosphorylation in which the

(1) RO-PO H + HOH'+ ROH + H-PO

enzyme acts as a phosphotransferase, does not involve the intermediate
formation of inorganic phosphate in transmission of the phosphoryl group
to the accepter:
(2) R0-P03H2 + XOH-+ ROH + X-OPOBH
This reaction, which results in phosphoric ester synthesis, does not
require the presence of adenosine triphosphate (ATP) or other high energy

phosphates (Morton, 1958). Note that reaction (1) is really a special

case of reaction (2).

5

It has been generally assumed that reaction (1) characterizes the
action of alkaline phosphatase in its natural environment, and all methods
of estimating the enzyme are based on this premise. Whether the enzyme
predominantly catalyzes reaction (1) or (2) appears to depend upon compe-
tition between water and other hydroxyl containing compounds for sites at
the surface of the enzyme—donor-complex. Nothing definite is known as yet
of the substrate.binding groups at the active center of the enzyme.

Phosphatase, in splitting off the phosphate group from p-nitrophenyl
phosphate, liberates p-nitrophenol which is yellow in alkaline solution
and therefore serves as an indicator. Thus, alkaline phosphatase activity
is measured by the intensity of yellow color in alkaline media in the
presence of a specific substrate. Activity units are arbitrarily defined
on the basis of method and conversion charts are available for conversion
of either Bodansky.or KingeArmstrong units to International units (see
Appendix).

The normal values for plasma alkaline phosphatase are 2.0 to 4.5
Bodansky units/d1. in adults, and 3.5 to 11.0 Bodansky units/d1. for
children. Since alkaline phosphatase is normally excreted by the liver,
the values are increased in obstructive jaundice. In a purely hemolytic
jaundice there is usually no rise. Unfortunately, various other factors,
may affect phosphatase activity so that the results of this test must be

correlated with clinical findings and with other tests (Harper, 1965).

HISTORICAL REVIEW

Robinson's discovery of alkaline phosphatase in bone in 1923, and
his theory of the role of this enzyme in bone formation, stimulated investi-
gation of the relationship of-skeletal disease to the serum alkaline phos-
phatase. Markedly increased alkaline phosphatase activity in the serum
has been demonstrated in Paget's disease, hyperparathyroidism (osteitis.
fibrosa), rickets, osteosarcoma and carcinoma with widespread metastases,
just to mention a few (Gutman, 1936; Bodansky and Jaffe, 1934).

A second category of diseases, involving the hepatobiliary system,
was found to be associated with increased phosphatase activity in.the
plasma by Roberts (1933). He noted augmented values in patients with
jaundice due to obstruction of the extrahepatic biliary tract and normal
or slightly elevated levels in patients with catarrhal, infective, toxic
and hemolytic jaundice. Roberts therefore proposed use of the determina-
tion of alkaline phosphatase to distinguish obstructive from other forms
of jaundice. However, Bodansky and Jaffe (1933) found extensive overlapping
of total alkaline phosphatase results in cases of hepatogenous jaundice
and suggested discontinuing the use of this determination to differentiate
obstructive from hepatogenous jaundice. The level of total serum alkaline
phosphatase activity was later found to be a more useful index of certain.
forms of hepatobiliary disease than was first appreciated, since it reflects
obstruction not only of the extrahepatic biliary tract, with or without
oVert jaundice, but also obstruction of the intrahepatic biliary tract,
with or without jaundice. Some metastatic carcinomas of the liver have

6

7
been seen wherein the alkaline phosphatase activity is increased in.
advance of hyperbilirubinemia (Meranze et al., 1938). It was concluded
from these early studies that the determination of serum alkaline phospha-
tase affords, in man, a sensitive criterion of the patency of the excre-
tory biliary channels, extrahepatic and intrahepatic (Gutman, 1940).

By 1940, investigation of the serum alkaline phosphatase had thus
revealed markedly increased levels of activity in 2 general categories
of human disease: disorders of the skeleton characterized by overactivity
of substantial numbers of osteoblasts, and disorders of the hepatobiliary
system, notably those characterized by obstruction of the extrahepatic
or intrahepatic biliary tract.

Alkaline phosphatase has been extensively used in diagnosis during
the past 3 decades, and there has been much discussion concerning the
origin of the serum enzyme in hepatobiliary disease. Bone appears to be
the principal source of alkaline phosphatase, and there is strong evidence
indicating that this enzyme is mainly excreted in the bile (Gutman, 1959).
Many other tissues, including the intestine, kidney and placenta, are
known to contain this enzyme (Bodansky, 1948; Ross et aZ., 1956). The
possibility remains that some other tissue might contribute to an increased
serum alkaline phosphatase activity.

Among the procedures which have been applied in attempts to identify
the tissue(s) of origin of the serumrenzyme activity are chromatographic
and electrophoretic techniques. These methods have led eventually to
recognition of its heterogeneity. The existence of serum alkaline phos-
phatase in more than one form was first indicated by paper electrophoresis
by Baker and Pellegrino in.l954. After starch-gel electrophoresis, activity
was extracted from 2 zones, one of which moves more slowly than the slow

az—globulin and the other slightly more slowly than the B-globulin,

8
(Kowlessar, 1958). Both zones show increased activity in liver disorders,
but there is an increase in the B-globulin region only in bone disease.
The test-paper method for visualizing the zones of activity was intro-
duced by Estborn in 1959. This has demonstrated a major band which
travels slightly more slowly than B-globulin and a second faint band
corresponding to what has been described as the prealbumin (acid al-
glycoprotein) zone.

The application of ion-exchange chromatographic techniques has also
shown that the human serum enzyme consists of at least 2 distinct come
ponents. Fahey, MoCoy and Goulian (1958) detected 2 peaks of activity,
both of which appear long before the single peak of acid phosphatase
activity, using chromatography on DEAE cellulose columns.

Quantitative studies with the aid of starch block electrophoresis:
also indicate that the major zone of the serum alkaline phosphatase
activity migrates with the az-globulins (Rosenberg, 1959), but Keiding
(1959) succeeded in resolving this into an "dz-component" migrating near

the a -globulin and the "B-component" located between the aZ—globulin

2

and the B-globulin. Both workers found a minor band in the a -globulin

1
region in the sera of patients with liver disease. Since the B<globulin-
fraction is considerably increased during childhood, and is particularly
prominent in the sera of patients with Paget's disease or bone metastases,
it would appear to be largely derived from bone. The al-component appears
to originate in the liver, and the finding that an appreciable fraction
of the alkaline phosphatase of the bile migrates as an (ll—globulin is
consistent with this conclusion. Further support for this view is pro-

vided by Cooke.and Zilva (1961), who reported a case in which extensive

metastases to the liver, but no bony metastases, were found at autopsy.

9
After starch-block electrophoresis, about 50% of the serum alkaline phos-

phatase activity was found in the a -peak.

1

Haiji and deJong (1963) applied agar-gel electrophoresis to the study
of the isoenzyme patterns of the alkaline phosphatases of human sera and
tissues. The results resembled those obtained by starch-block electro-
phoresis in showing 3 main bands of activity: Band I was slightly slower
and Band II slightly faster.than the az-globulins, while Band III had a
mobility between that of the 01- and az-globulins. A single band also
appeared between I and II, and was attributed to superimposing of these
2 bands as seen in patients with diseases involving both liver and bone.
This may also be detected by mixtures of serum containing individual
bands. There is general agreement in the interpretation of the results
of Haiji and deJong (1963) obtained by starchéblock electrophoresis:
Band I appears to be associated with bone diseases, while the faster
fractions, Bands II and II, predominate in.liver disease.

The improved resolution of protein fractions obtainable by starch-
gel electrophoresis confirmed the existence of these phosphatase components
(Markert and Moller,‘l959). Estborn (1959) showed that the principal
alkaline phosphatase in the serum of a patient with obstructive jaundice
due to cholelithiasis migrated as a distinct band between the a-globulin
and haptoglobin fractions. In certain other sera, this band was
accompanied by a weak band of activity occurring in the prealbumin (acid
“1) fraction. This component was also found in bile, but the principal
phosphatase of this fluid migrated somewhat more slowly than the main.serum
component. Estborn concluded that the serum alkaline phosphatase migrates
separately from the main serum protein fractions and that the association
with the a - and a

1 z-globulinsreported by investigators using starch grains

as support media is coincidental. Hodson, Latner and Raine (1962) came to

10
the same conclusion as a result of their observation that the positions
of the alkaline phosphatase bands relative to the major serum proteins may
be varied by using a discontinuous buffer system.

Numerous investigators have separated the alkaline phosphatase com-
ponents of human and animal serum and tissue by starch-gel electrophoreis
and, though the patterns obtained differ in detail, certain general con-
clusions may be drawn. The principal alkaline phosphatase band of liver
extract migrates close to the transferrin (B-globulin) fraction of serum;
minor bands in the B-lipoprotein and slow az-globulin zones are usually
detected. There may also be activity near the point of application.

The main band of kidney extracts occurs in the haptoglobin zone, while
minor bands may be seen in the B-lipoprotein and transferrin (B-globulin)

. regions. Most of the alkaline phosphatase of intestinal origin moves more
slowly than the main band of the liver enzyme; the slower fractions in the
B-lipoprotein region and near.the origin may be detected. Bone alkaline
phosphatase activity is usually found close to the B-globulin region.
Slower bands have been reported by some investigators (Moss, 1962).

Evidence that the slow-moving bands of alkaline phosphatase iso-
enzymes originate in the liver has been provided by elution of the enzymes
after starch-gel electrophoresis. The slow band of alkaline phosphatase
also exhibits considerable 5'nuc1eotidase activity, whereas the fraction
associated with the B-globulin is devoid of such activity (Kowlessar et
aZ., 1961). Since elevation of the serum 5'nuc1eotidase is highly specific
for liver diseases, these observations are consistent with the view that
the liver is the source of the slow components of the serum alkaline

‘phosphatase.~

11

Of 120 patients with a variety of diseases and having elevated serum
alkaline phosphatase activities, only 2 exhibited bands in other than the
B-globulin and haptoglobin I zones, and Boyer (1961) therefore concludes
that simple starch-gel electrophoresis is not likely to be useful as a
diagnostic tool. Hodson at al. (1962) point out that serum isoenzyme
separation might aid in the differential diagnosis of jaundice; for cases
of obstructive jaundice they noted a doublet of phosphatase activity near
the origin which is less prominent in sera from.patients with hepatocellular
disease.

Disagreement remains concerning the origin of the alkaline phosphatase.
in normal human serum. Most of the activity occurs close to the B-globulin
region, a fraction attributed by Kowlessar (1959) to bone phosphatase and
by Hodson (1962) to the liver enzyme. Chiandussi at al. (1962) conclude
that it would be of either hepatic or osseous origin.

A fourth fraction in cord blood, migrating with the_B-globulins, has
been reported by Chiandussi at al. (1962), who also found a similar band
in placental extracts. Boyer (1961), while concurring with this finding
in cord blood, also found a fourth fraction in serum from.women in labor.
The main.band is similar to that of bone extract, but different from.that
in the placenta and liver.

Quantitation of the isoenzyme fractions remains a difficult task
because of overlapping of fraction patterns. Nevertheless, utilizing an.
established electrophoretic technique for isoenzyme fractionation, with
only minor changes, the results indicate that normal levels previously
established for infants may not be valid when hyperbilirubinemia is

present.

MATERIALS AND METHODS

Source of Specimens
Blood from.the fetal-maternal cord was obtained at the time of
delivery by one of the staff obstetricians. The cord was cut and a 10~ml.
sample collected from the fetal side. All specimens were centrifuged
at 2000 rpm for 10 minutes when received and the resultant serum removed
and separated into aliquots of 0.5 to 1.0 ml. each. The labelled samples
were placed in the refrigerator (4 to 6 C.) until electrophoresis was

carried out.
Total Alkaline Phosphatase

Mgthgg, Dilute 1 drop (0.04 ml.) of substrate concentrate with 1.0 ml.
of water and warm to 37 C. Add 0.1 ml. of serum and mix. After exactly
20 minutes add 5.0 ml. of color stabilizer and read the absorbance at
550 nm against a reagent blank without serum. Determine the alkaline

phosphatase activity from the standard curve.

Standardization.~ Dilute 1.0 ml. of the stock phenolphthalein standard

to 50.0 ml. with water in_a volumetric flask. To 1, 2, 3, and 4 ml. of
working standard (0.05 umole/ml.) and 5.14, 4.14, 3.14, and 2.14 ml. of
color stabilizer, respectively, mix and read the absorbance at 550 nm.
Results are expressed in International units. The unit is defined as the
amount of enzyme that will liberate l umole of product in 1 minute. The

above standards are equivalent to 25, 50, 75, and 100 U., respectively.

12

13
The precision of the total alkaline phosphatase method as reported by
Babson-et al. (1966) was 40.3 to 41.4 U., with a mean of 40.9 U. The

coefficient of variation was 0.8%..
Alkaline Phosphatase Isoenzymes - Electrophoresis

lggpgents and apparatus
1. VElectrophoresis cell*
2. Power Supply (Beckman Duo—stat)**
3. Cellulose acetate strips, 1 x 6-1/2 in.*
4. Sample applicator (Beckman 1 in.)**

5. Barbital buffer, pH 8.6, ionic strength 0.025

Method. Cold barbital buffer (700 ml.) is poured into the cell chambers
and the chambers equilibrated. Cellulose acetate strips are saturated
with buffer in a small tray, removed, laid on blotter paper and blotted

to near dryness.

Sapple application. A 0.01-m1. pipet*** calibrated at 0.01, 0.008,
and 0.006 is used to deliver 0.006 ml. of serum to the edge of the applicator,
which is then brought into contact with the moist cellulose acetate strip
resting on the blotter paper in a horizontal straight line about 1/2 in.
cathodic to the center of the strip. Instant absorption occurs by capil-

lary action in a narrow band of serum across the strip. The strips are

 

*

Gelman Instrument .00., Ann Arbor, Michigan.
**

Beckman Instruments, Palo Alto, California.

***
‘ Spinco Division, Beckman Instruments, Palo Alto, California.

l4
placed into the cell, and the magnetic rubber gripping devices are then
attached, which act as a tension device holding the strips in place. The
cell cover is replaced and a voltage of 17.5 v/cm. (of cellulose acetate
strip length) is applied for 40 min. at 4 to 6 C.--convenient1y in.a
refrigerator. [Note: This experiment was conducted by placing the

Gelman cell on a crushed ice bath.]

Detection

Reagents
l. Buffered phenolphthalein monophosphate substrate
(see Appendix)

2. Color stabilizer

Method. Dilute 0.2 ml. of the phenolphthalein monophosphate sub-
strate concentration with 2.0 ml. of water. This is sufficient quantity
to saturate 3 cellulose acetate strips. Pour the diluted substrate into
a shallow tray and immerse 3 clean strips into the solution. Using forceps,
remove the strips from the tray, allowing excess solution to drip off, and
lay them on a clean, flat, glass plate approximately 10 x 5 in. For con-
servation of cellulose acetate strips, the strips may be cut in half and
placed on a 1 x 3 in. glass hematology slide. Disconnect the current to
the cell and remove the strips containing the separated specimens. With
care to avoid any air pockets from forming, superimpose them onto the
strips containing the substrate. Place the glass plate or slides contain-
ing the specimens into a moist atmosphere, such as a large plastic box
containing a few milliliters of water. Incubate the specimens in a 37 C.

bacteriologic incubator for 2 hours.

15

Elution of activity. The cellulose acetate strips are removed from
the incubator and the isoenzyme bands of activity, which may or may not
be completely visible, are cut at 1 cm. intervals from the origin to the
marker band of albuminebound bilirubin. The segments are placed into 13
x 100 mm. tubes to whiCh 3.0 ml. of the color stabilizer has been added.
Mix the tubes either by inversion for several minutes or by using a glass
rod, until all the visible color has been eluted from the strip. A blank
is prepared by cutting a segment of the strip free of enzyme activity and
placing it into 3.0 ml. color stabilizer. The alkaline phosphatase
activity is read in 12.x 75 mm. cuvettes onta spectrophotometer at 550
nm against the reagent blank. Conversion of the %T (transmission) read-
ings to O.D. (optical density) was made and the results were read from a
curve prepared under similar conditions. A semiquantitative estimate of
the percentage of enzyme activity attributed to each isoenzyme can be
calculated by the following equation:~

absorbancejof isoenzyme-reagent blank x 100 a 7

total absorbance-reagent blank activity

Standard curve. A standard curve was prepared for use in this experiment.
*
Dilutions and combinations of Versatol E and EN were prepared in the

following manner:

 

*
Commercial control sera from General Diagnostics Division, Warner-
Chilcotte, Morris Plains, New Jersey.

16

 

 

Combination Unit Value 9‘2;
1. Ver. EN 5.8 0.029
2. E + EN + 1 vol. Saline. 8.5 0.041
3. (3E + 1 EN) + 1 vol. Saline 11.3 0.061
4. l E + 1 saline 14.1 0.066
5. E + EN 17.1 0.071
6. 3E + 1 EN 22.7 0.076
7. Ver. E 28.3 0.089

EN - 5.8 International units (normal)
E - 28.3 International units (abnormal)
The curve was plotted to a straignt line and utilized in the calcula-
tion of the alkaline phosphatase units described in this thesis. The.
dilutions prepared were subjected to electrophoresis in the same manner.

as the serums and then eluted to obtain the optical density readings listed.

Experiment

Prior to the electrophoretic analysis of the cord bloods, several
parameters were tested. Initially, storage time and temperature.were.
determined. Sera from 5 patients were analyzed by a manual method for
determining alkaline phosphatase activity. Aliquots were prepared, one
set frozen at -20 C. and the second set stored at 4 C. Daily for 5 days,
1 aliquot from each thermal store was analyzed for total alkaline phospha-
tase activity. No appreciable difference was detected in the total values
over the 5-day period. However, when electrophoresis was carried out on
each daily sample, results obtained after the third day showed decreasing
values in the isoenzyme fractions of the bone and liver. The use of fresh
sera for isoenzyme fraction was considered an important factor for the

data collection to be relevant.

l7

Electrophoresis was then carried out on sera from patients with
elevated levels of alkaline phosphatase, predetermined from manual testing
to be either osseous or hepatic in origin. Following electrophoretic
analysis, elution of the isoenzyme activity was compared to the values
ascertained from the manual thermolabile technique. Mixtures of sera
from patients with metastatic bone tumors and obstructive jaundice were
analyzed electrophoretically, the 2 isoenzyme bands were eluted and again
compared to manual techniques. The results were comparable once the
operation of the electrophoretic equipment was mastered.

The length of time for optimal migration of the isoenzyme activity
was determined by the staining of several different cellulose acetate
strips for protein. Protein migration was well defined at 40 minutes of
electrophoresis time and this time‘was used throughout the experiment.
The hepatic and osseous_a1kaline phosphatase isoenzyme.bands were well
defined with little or no visible overlapping at 40 minutes when the 2
fractions were present in mixed sera.

The temperature 0f the barbital buffer during electrophoresis was a
critical factor, as was the number of times the buffer was charged. It
was found that a 2 C. rise in.the buffer during electrophoresis destroyed
a considerable amount of the activity of the osseous fraction as well as
causing burning effects on the cellulose acetate strip itself.

Romel et al. (1968) suggest refrigeration of the cell but as this
was not feasible in our situation, an alternative was constructed. A
styrofoam.box, with 1-in. walls and 3/4 in. cover was cut down so that
the Gelman cell would fit snugly inside. Space beneath the cell, about
4 in., was filled with crushed ice. This device kept the precooled

buffer at 4 C. to 1 C. throughout the period of electrophoretic analysis.

18
The.temperature was checked before.and after each analysis until known
to be stable, thereafter checked periodically.

The number of times the buffer could be charged was determined by
repeated use of the same serum. At each new application of the current,
the electrical terminals were reversed so migration was - to +, +‘to -,
etc.' After 2 charges, it was noted that the buffer failed to conduct
adequately, resulting in a poor migration pattern. This experiment was
conducted by using the same buffer twice only. Fresh buffer was used at
the beginning of each day analysis was carried out.

Refrigeration of the Gelman cell for a period of one-half hour
before use made it possible to begin electrophoresis 5 minutes after the
cell had been transferred to the "cooling box" and the cold buffer had
equilibrated.

It should be noted that bilirubin bound to albumin serves as a good
migration marker, and, as such, was a check on electrophoresis. time. It
was found that this marker band had migrated 3.5 to 4.0 cm. from the
point of origin after 40 minutes. Extremely high bilirubin levels did
cause drag of the marker band but generally created no problems in the
elution method.‘ However, if the marker drag is greater than 0.5 cm.,
complete elution of the hepatic fraction is difficult.

Serum.obtained from submitted cord samples were separated into 3
aliquots of 0.5 to 1.0 m1. (when blood was obtained in microcapillary
tubes, 0.1 m1. of serum was aliquoted). Fresh serum was subjected to
electrophoresis initially. A 0.006 ml. sample was placed on the cellulose
acetate strip as previously described and electrophoresis was carried out
for 40 min. in the Gelman cell. A standard electrophoresis dye was used
‘with each set of sera processed to check the electrode function and

direction of migration.

19

It was found that the substrate could be adequately applied to a
1px 3 in. cellulose acetate strip for the incubation phase (cut a regular
strip in half). The substrate-soaked strips were then placed on a 1 x 3
in. laboratory glass slide. The processed strip was then cut 1 cm.
cathodic to the origin placement and 2 cm. anodic to the marker band and
this was superimposed on the 1 x 3 in. substrate strip. These strips were
then placed in a 37 C. bacteriological incubator for 2 hours.

During incubation of the first samples, one of the aliquots of the
original specimen was inactivated for 15 minutes at 56 C., followed
immediately by immersion in an ice bath. After cooling, this sample
was subjected to electrophoresis by the established procedure. The
electrical terminals were reversed and electrophoretic analysis was
carried out for 40 min.

During incubation of the second sample, a third aliquot was inacti-
vated for 30 min. at 56 C. and processed in the manner just described.

Following the 2-hour incubation phases, each set of strips was cut
at 1-cm. intervals from the origin to the marker band and elution was
carried out in a 16 x 100 mm. tube containing 3.0 ml. color stabilizer.
The blank was prepared as previously described. The results of elution
were read at 550 nm. on a Coleman Junior 11 spectrophotometer using
12 x 75 mm. cuvettes. The elution of the total color generally took
about 15 minutes. The ZT readings were converted to O.D. values and
the alkaline phosphatase units read directly from the standard curve.

The second and third aliquots were cut at 1-cm. intervals only for
convenience of elution as only a total alkaline phosphatase value was

recorded.

20

The cut segments of the original, fresh sample represent the 3

isoenzyme fractions in this manner:

0-1 cm. placental fraction
1-2 cm. osseous fraction
2-3 cm. hepatic fraction

RESULTS

The results obtained are listed on the following tables.

Table 1. Electrophoretic analysis of alkaline phosphatase isoenzymes in.
"normal" infants (results in International units)

 

 

 

 

 

 

Total Ii-f Liver i-_-_—T§;;;-_-
Alkaline (unstable) (unstable) Placental
Specimen ghgsphatase. 39:; 56° 15: 56° (gtable)
1 35.5 2.3 31.4 1.7
2 28.8 2.1 26.1 0.8
3 29.1 1.5 25.2 1.7
4 24.3 0.8 22.2 0.8
5 42.2 2.2 38.5 1.9
6 37.5 1.9 35.2 0.2
7 29.3 1.2 27.2 2.0
8 43.6 1.6 38.0 2.5
9 34.7 2.3 29.9 1.9
10 41.0 2.0 37.0 2.0
11 39.3 1.3 35.2 2.9
12 34.0 1.2 30.0 1.2
13 42.6 2.2 38.0 2.0
14 35.7 2.3 32.4 1.9
15 36.2 1.0 33.6 1.8
16 28.4 1.3 25.3 1.9
17 33.8 1.1 30.6 - 1.9
18 37.7 1.3 33.8 2.3

21

Table l (cont'd.)

Total

2

2

 

 

 

 

 

 

Liver Bone

Alkaline (unstable) (unstable) Placental

Specimen Phosphatase 30' 56° 15' 56° (stable)
19 31.2 0.9 28.4 1.7
20 42.1' 2.5 38.6 1.2
21' 39.3 1.5 34.3 4.1
22 36.7 1.7 33.5 1.5
23 29.6 1.2 27.2 1.4
24 41.9 2.2 36.9 2.6
25 36.3 0.9 33.6 1.8
26 33.5 1.5 29.8 2.1

 

23

Table 2. Electrophoretic analysis of alkaline phosphatase isoenzymes in
infants with an ABC incompatibility (results in International
units)

 

Total
Alkaline
Specimen Phosphatase Liver Bone, Placental
1 16.8 2.3 13.1 1.4
2 26.2 4.4 21.0 0.8
3 33.8 2.7 28.2 1.9
4 26.1 1.9 21.5 2.8
5- 25.6 1.8 22.0 1.9
6 29.1 2.4 24.9 1.8
7 27.5 1.9 23.8 1.8
8 21.6 1.2 17.6 2.8
9 18.9 2.5 16.0 0.4
10 24.3 0.5 22.2 1.6
11 26.1 1.8 21.9 2.4
12 25.6 1.8 22.0 1.8
13 28.8 1.6 25.4 1.8
14 24.4 1.3 21.6 1.5
15 24.6 1.8 22.0 0.8
16 24.3 0.5 22.2 1.6

17 19.6 0.9 19.6 4.0

 

24

Table 3. Electrophoretic analysis of alkaline phosphatase isoenzymes in
infants with an Rh incompatibility (results in International

 

 

units)
Total
Alkaline
Specimen Phosphgtase Liver Bone Placental

1 28.6 3.9 22.8 1.9
2 41.9 2.2 35.9 3.8
3 19.1 0.2 13.3 5.6
4 18.8 0.6 16.8 1.4

5 1506 008 1009‘ 309

 

Table 4. Tabulation of the mean and standard deviation (S.D.) of alkaline

phosphatase fractions in "normal" and "abnormal" newborns

 

W

 

 

 

 

 

 

 

Total
Alkaline
Phosphatase. Liver Bone Placental
Nppppl cases: N - 26
Mean 35.5 1.7 32.0 1.8
S.D.- 5.2 0.6 4.6 0.6
Coef. Var. 14.6 40.0 14.6 37.4
ABO Incoppatibilities: N -;1__
Mean 24.9 1.8 21.4 1.8
S.D. 4.0 0.9 3.5 0.8
Coef. Var. 16.4 49.9 16.4 46.3
Rh Incompatibilities:' N;- 5
Mean 24.8 1.5 19.9 3.3
S.D. 10.7 1.5 9.9 1.6
Coef. Var. 43.2 98.6 50.0 51.0

 

Note: Mean and S.D. expressed in International units.

26

 

 

 

Table 5. Mean values obtained for "normal" and "abnormal" newborns and
the results of the application of the t-test for unpaired data

Fraction Normal Abnormal
Liver 1.7 1.7
Bone 32.0 21.1
Placenta 1.8 2.1
Total 35.5 25.0
t-test
Liver t a 0.41 P - ~<50 2252
Bone t a 7.9 P - 0.1%
Placenta t - 0.41 P - <50 >-25%
Total t = 7.13 P - 0.1%

 

Fractional values expressed in International units.

DISCUSSION

The alkaline phosphatase levels in jaundiced infants are consistently
either at the low end of the normal range or below. This decrease is
due almost entirely to a low concentration of the isoenzyme fraction derived
from bone. Since there is no reason to believe that there is stimulation
of hepatic excretory processes by jaundice, it would appear that bilirubin
in some way inhibits the activity of osteoblasts in the formation of
alkaline phosphatase, or that hyperbilirubinemia and decreased phosphatase
levels have a common cause. Consider, then, the possibility that osteo—
blasts possess surface antigens similar or identical to those present on
erythrocytes and that, when there is sufficient antibody derived from the
maternal side to destroy many erythrocytes, a significant number of
osteoblasts are also damaged. undoubtedly the quantitative relationships
between the concentrations of various specific protein types are important
in this concept. Thus, we must picture the osteoblasts aligned on spicules
of osteoid but separated from.the plasma compartment by interstitial space,
extracellular fluid, and the endothelial lining of the capillary wall
(Figure.l). If detectable amounts of phosphatase arising from osteoblasts
are present in plasma, the concentration of this protein immediately
adjacent to the osteoblasts must be relatively high. However, the fact
that detectable levels of enzyme do appear in the plasma establishes.the
ability of proteins to pass from osteoblasts to the plasma compartment.
With a steady influx of antibody protein into the fetal plasma from the
maternal circulation, inevitably some of this protein, as well as complement,

27

28

osteoblasts
layered

endothelium (5?

interstitial
space

 

 

osteocyte

 

osteoid

erythrocyte (:>

 

Figure 1. A diagram of the proposed site of action of the maternal
antibodies on the neonatal osteoblasts.

29
must reach the osteoblasts in sufficient concentration to interfere with
the production of alkaline phosphatase.'

During the analysis of the data, it was found that the liver fraction
of alkaline phosphatase was-unchanged when the 2 groups were compared.
This finding was not expected, since the project was designed to specifi-
cally look at the enzyme maturation of alkaline phosphatase in the liver.
As is stated above, the unexpected findings in the osseous fraction
changed the direction of the conclusions that were drawn. One can con-
clude, however, that the effect of hyperbilirubinemia in the neonate is
unrelated to maturation of the liver alkaline phosphatase isoenzyme.

The statistics noted for placental alkaline phosphatase were not a
primary consideration at this time, but were calculated to complete the

data.

SUMMARY AND CONCLUSIONS

Measurements of serum isoenzyme.1evels of alkaline phosphatase
were made on 2 groups of jaundiced infants: (1) those with transient
"phsyiological" jaundice and (2) those with jaundice due to fetal—
maternal incompatibility leading to hemolysis. Assuming that relatively
few of those with physiologic jaundice would manifest some degree of
immaturity of the hepatic excretory enzyme systems and that most of.
those with hemolytic jaundice would show varying degrees of the same
immaturity, the experiment was designed to reveal any association of
immature enzyme systems, glucuronyl transferase and phosphatase in this
instance.

The data reveal no associated decrease in the hepatic fraction of
phosphatase enzymes. However, an unexpected decrease in the osseous
fraction of alkaline phosphatase was encountered in the infants with
hemolysis. The hypothesis is offered that osteoblasts have surface
antigens in common with erythrocytes and that sufficient complement and
hemolytic antibodies reach the osteoblasts to produce an appreciable

drop in their output of alkaline phosphatase.

3O

BIBLIOGRAPHY

BIBLIOGRAPHY

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159.

Babson, A. L., Greeley, S. J., Coleman, C. M., and Phillips, G. E.:
Phenolphthalein monophosphate.as a substrate for serum alkaline.
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Baker, R. W. R., and Pellegrino, C.: The separation and detection.of serum
enzymes by paper electrophoresis. Scand. J. Clin. and Lab. Invest.,
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Bodansky, A., and Jaffe, H. L.: Phosphatase studies. IV. Serum phos-
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Bodansky, A., and Jaffe, H. L.: Phosphatase studies. III. Serum phos-
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Arch. Int. Med., 54, (1934): 88.

Bodansky, A.: Nonéosseous origins of serum phosphatase:- The liver.
Enzymol., 3, (1937): 258.

Bodansky, 0.: Are the phosphatases.of bone, kidney, intestine and serum
identical?: The use of bile acids in their differentiation. J.
Biol. Chem., 118, (1937): 341.

Bodansky, 0.: The inhibitory effects of L-alanine, L-glutamic acid, L-
lysine and L-histidine on the activity of intestinal, bone and
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Boyer, S. H.: Alkaline phosphatase in human sera and placenta. Science,
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Brown, A. K.: Bilirubin metabolism with special reference to neonatal

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Claireau, A. E.: Neonatal hyperbilirubinemia. Brit. Med. J., l, (1960):
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31

32

Coodley, E. L.: Diagnostic Enzymolggy. I. Enzymes in hepatic disease.
Lea and Febiger Publishers, Philadelphia (1970).

Cook, K. B., and Zilva, J. F.: Serum alkaline phosphatase fractionation
as an.aid to diagnosis. J. Clin. Path., 14, (1961): 500.

Edsall, J. F. (ed.): Enzymes and Enzyme Systems. Harvard Press (1951).

Estborn, B.: Visualization of acid and alkaline phosphatase after starch-
gel electrophoresis of seminal plasma, serum and bile. Nature,
London, 184, (1959): 1636.

Fahey, J. L., McCoy, P. F., and Goulian, M.: Chromatography of serum
proteins in normal and pathologic sera: Alkaline and acid phos-
phatase.~ J. Clin. Invest., 37, (1958): 272.

Gutman, A. B., Tyson, T. L., and Gutman, E. B.: Serum calcium, inorganic
phosphorus and phosphatase activity in hyperparathyroidism, Paget's
disease, multiple myeloma and neoplastic disease of the bones.
Arch. Int. Med., 57, (1936): 379.

Gutman, A. B., Olson, K. B., Gutman, E. B., and Flood, R. A.: Effect
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Haije, W. G., and deJong, M.: Isa-enzyme patterns of serum alkaline
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Harper, H. A.: Review of Physiological Chemistry, 10th ed., Lange Medical
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Keiding, N. R.: Differentiation into three fractions of the serum alkaline
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33

Markert, C. L., and Mo11er, F.: Multiple forms of enzymes: Tissue,

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Romel, W. C. ,LaMancusa, S. J., and DuFrene, J. K.: Detection of serum
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APPENDIX

APPENDIX

Total Alkaline Phosphatase Reagents~

1. Buffered substrate concentrate: Dissolve 65 mM phenophthalein
monophosphate in 7.8 M 2-amino-2-methy1propanol buffer at pH
10.15. The concentrate is stable-under refrigeration and
should be warmed to room temperature before use.

2. Color stabilizer: Disodium hydrogen phosphate-trisodium
phosphate, 0.1 M, at pH 11.2.

3. Phenolphthalein.stock standard, 2.5 mM: Dissolve 79.6 mg.
of phenolphthalein in 50 ml. of alcohol in a lOO-ml. volumetric
flask and dilute to lOO-ml. with water. The solution is stable
at room temperature.

4. Barbital buffer: Dissolve 5.12 gms. sodium barbital and
0.92 gms. of barbituric acid in.a l-liter volumetric flask.
Dilute to 1 liter.

Note: Barbital buffer, color stabilizer and the substrate
can be purchased in pre-pack form from General Diag-
nosticsAWarner Chilcotte, Morris Plains, New Jersey.

Conversion of International Units of Alkaline Phosphatase to:
l. ‘Bodansky units - multiply I.U. by 0.12.

2. KingeArmstrong units - multiply I.U. by 0.36.
3.‘ Shinowara-Jones-Reinhart units - multiply I.U. by 0.18.

Heat Stability:

1. Osseous fraction is destroyed by exposure to 56 C. for 15 minutes.
2. Liver fraction is destroyed by exposure to 56 C. for 30 minutes.
3. Placental fraction stable.

34

VITA

The-author was born in Parnell, Michigan, on April 15, 1938. She
lived there until graduation from St. Btrick's High School in 1956.

In September of that year, she entered Mercy College of Detroit, on a
Silverman Honors Scholarship, graduating with a 3.8. degree in 1960 after
completion of a 12-month internship for Medical Technologists at Mt.
Carmel Mercy Hospital, Detroit. Later that same.year, she became certi-
fied with the Registry of Medical Technologists of the American Society
of Clinical Pathologists.

In June 1960 she began working at Ferguson-Droste-Ferguson Hospital
and ProctologyClinic in Grand Rapids, Michigan. She remained there for
5 years, 3 years ‘of which she was Chief Technologist of the laboratory.

In July 1965 she moved to. AnnArbor, Michigan, where she worked as
supervisor of hematology, later becoming supervisor of the Blood Bank-
at St. Joseph Mercy Hospital.

In September 1966 she-entered the AABB approved school at Michigan ..
Comnmnity Blood Center in Detroit, Michigan, for a year of training in
Blood Banking. She was certified by AABB and the American Society of
ClinicalPathologists . in April 1968.

She was employed atEdward W. Sparrow Hospital, Lansing, Michigan,
in Septenber 1967, as Supervisor of Blood Bank and Immunoserology, the
position she currentlyholds.

She entered Michigan State University in September 1968 in the
Clinical Laboratory Scienceprogram, Department of Pathology. She worked

35

36
full time at E. W. Sparrow Hospital during the graduate program.
The author is an active.member of the Lansing area, Michigan, and
American Society of Medical Technologists, and the Michigan.and American

Association of Blood Banks.