Download Isoenzymes in Clinical Diagnosis

SPECIAL ARTICLE
Isoenzymes
in
Clinical Diagnosis
By THEODORE L. GOODFRIEND, M.D.,
AND
NATHAN 0. KAPLAN, PH.D.
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The acid and alkaline phosphatases are not
usually designated as isoenzymes. This term
was first applied to enzymes that catalyzed
the same reaction but differed from one another in electrophoretic or chromatographic properties. For the purposes of this review, the
definition of isoenzymes will be broadened to
include all sets of enzymes that catalyze a
given reaction, regardless of the nature of
the differences among them. By this definition,
acid and alkaline phosphatases are isoenzymes,
and the recently described electrophoretic
forms within each of these groups are also
SERUM ENZYMES are useful tools for determining the location and severity of
many diseases. They are protein catalysts,
some of which enter the serum from damaged
tissues. Because they are catalysts, they are
more easily detected than many other substances. Amylase released from the diseased
pancreas,1 and phosphatase released from
several tissues2 have been studied for many
years and have established the usefulness of
serum enzyme tests.
Unfortunately, many of the best studied
and most easily detected enzymes occur in
more than one organ. Furthermore, some organs like liver and skeletal muscle contain
high concentrations of many enzymes and
frequently cause confusion in diagnosis
based on enzymes.
In one respect, however, the wide distribution of some enzymes is illusory: the enzyme
activity is widespread, but the specific protein
catalyst may vary from tissue to tissue. This
was recognized early in the case of phosphatases.3 Bone, prostate, and red cells are
rich in proteins catalyzing the hydrolysis of
phosphates, but the major phosphatases from
these tissues differ in pH optima and susceptibility to inhibition by a variety of chemicals.
Different enzyme molecules that catalyze the
same reaction are called "isoenzymes," 'isozymes," or "multiple molecular forms." Their
discovery has encouraged further searches for
organ-specific catalytic proteins.
isoenzymes.
Characteristics of Isoenzymes
Enzymes that catalyze the same reaction
may differ from one another in many ways,
ranging from small variations in secondary
structure to broad differences in amino acid
sequence and molecular weight. These criteria
are listed in table 1. At one extreme are enzymes with marked differences in structure,
but a common substrate. The esterases13 and
the peptidases'4 are probably in this class. At
the other extreme are molecules that are identical in all respects but their degree of denaturation. Separatory procedures are now so
sensitive that multiple forms of enzymes may
appear, which merely reflect artifacts of preparation or handling of the specimens. Relatively minor manipulations may introduce differences caused by the folding of enzyme
protein chains,4 the aggregation of chains into
polymers,5 the addition or removal of lipids,11
bound metals,15 or deamidation of carboxyl
amide groups.8 For clinical studies, the
isoenzymes with the greatest value are those
that differ in a fundamental way, which per-
From the Graduate Department of Biochemistry,
Brandeis University, Waltham, Massachusetts. Publication no. 393.
Supported in part by grants from the National Institutes of Health, U. S. Public Health Service, and
the National Science Foundation.
1010
Circ0lation, Volume XXXII, December 1965
ISOENZYMES IN CLINICAL DIAGNOSIS
1011
Table 1
Diferences in Isoenzymes
A. Physicochemical
1. Differences in secondary and tertiary structure, (folding of polypeptide chains), e.g.,
ref. 4
2. Different degrees of polymerization to dimers, tetramers, etc.5
B. Immunochemical
1. Different reactivity with specific antibodies.6
C. Chemical
1. Variations in degree of deamidation of carboxylamide groups or acetylation of amino
groups.7, 8
2. Variable combinations with carrier proteins, carbohydrates, coenzymes, prosthetic
groups, or lipid.9-11
3. Different degrees of activation or inactivation by hydrolytic cleavage of terminal
peptides, oxidation or reduction of coenzyme or sulfhydryl groups.
4. Varying degrees of amino acid differences.12
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sists despite the influence of serum contaminants, storage, or analytic technics.
Detection of Isoenzymes
Two groups of phosphatase were originally
detected by their different pH optima and
called "acid" and "alkaline" phosphatase. Further "isoenzymes" of phosphatase were differentiated by susceptibility to inhibition by
tartrate and other chemicals.'6 Different pepsins were separated by differences in solubility.17 Since these early examples, methods of
differentiation have increased. The most widely used are technics of separation by electrophoresis or chromatography. The basic methods of isoenzyme detection and measurement
are listed in table 2.
Figure 1A is an example of lactic dehydrogenase isoenzymes (LDH), separated by electrophoresis and detected by a staining reaction
specific for this enzyme. Figure lB shows an
analogous pattern for creatine kinase. Once
separated, the isoenzymes may prove to have
different catalytic properties. For example, the
muscle and heart varieties of LDH behave
differently toward various concentrations of
their common substrate, as shown in figure
2. These differences, like different susceptibility to pH, inhibitors, and specific antibodies,
may be utilized for detecting and measuring
the various enzyme forms.
The isoenzymes that are currently most convenient for clinical diagnosis are those which
can be differentiated by simple assays using
Circulation, Volume XXXII, December 1965
small amounts of enzyme, and various conditions of heat treatment, pH, substrate concentration, or inhibitors. Such assays do not
require preliminary separation of the proteins by chromatography, etc. The heart and
muscle forms of LDH are examples of isoenzymes detectable by such chemical properties. In fact, the demonstration of experimental data like that shown in figure 2 is an
indication that such methods are applicable.
These curves indicate at a glance the conditions of assay that will differentiate the two
forms (shown by the vertical dotted lines),
one condition measures both forms equally
well, the second condition specifically inhibits
one of the two forms. Such an assay is precise and quantitative. If more than two isoenzymes are present, however, this type of
test may not give as complete a picture of the
spectrum of isoenzymes as electrophoresis or
chromatography. Finally it may be possible to adapt separatory procedures to a sort
of chemical assay. For example, isoenzymes
that adhere relatively firmly to materials used
in chromatography can be removed from assay mixtures by the "batch" addition of gel to
the specimen. In this way, the gel is used
instead of a chemical inhibitor.
Origin of Isoenzymes
The isoenzymes that differ in amino acid
composition, such as the isoenzymes of LDH,
probably represent the results of ancestral
mutation and gene duplication: a single gene
1012
GOODFRIEND. KAPLAN
Table 2
Alethods for Detection arnd Measunreinent of lshoeiZyjm es
A. 1b1wsicochemical
I. Separation by electtropborcsis.ls.
2. Separation
b)y chromattog:aph)l1yA
B. Ilalmmllwocl)emical
l. Combination vitl, 01. inhliil)ition 1by specific inti1bodies. 211
C. Chemical
1. Bate of reaiction und(ler vatriomus condlition.s of p112 temperiture,21 inhibitors,1" cociazyme
analogtue's, or substrate concentration.2
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corresponding to a giveni enzyme gave rise to
two or more genes and two or more different
enzymes. Those isoenzymes that differ in
prosthetic groups, secondary structture, or
state of polymerization may have arisen by
other routes. In some cases, further evolution or environmental influences caused separate genes to become expressed to varying
degrees in different organs. This resuilted in the
organ-specific isoenizyme patterns under discussion. There are other instances, niotably
malic.' and TPN-linked isocitric dehydrogenases$2 in whiclh the isoenzymes are found in
many tissues l)ut are localized in different subcellular compartments, such as mitochondria
anid cytoplasm.
Genetic and evolutionary factors havce
given rise to differences in other well-studied
proteins uvhich share a common ftinction, such
as hemoglobins, haptoglobins, and gamma
globulins. Another exam-ple is the hormonepair oxytocin aind vasopressin, xhich share
common properties and probably arose from
a single ancestor like the vasotocin of loNver
forms. It has lonig been recognized that some
ssuch "iso" proteins vary from species to species, from race to race, or from person to person. The coexistence of mutltiple forms of enzymes or other proteins within the same
individual but localized in various organs is
the featuire that makes them useful in clinical
diagnosis.
The existence of two different genes for
fuinctionally related enzymes may give rise to
more than tu0o isoenizymes. This resuilts from
the fact that some enzy mes are composed of
A
S
LACTIC DEHYDROGENASE
LDH
ISOENZYMES
CREATINE KINASE
M4 M3}H
M2H2 MMH3 H
MHMH2MHH4
BRAI N
BRAIN
I
HEART
HEART
SKELETAL
MUSCLE
'Ro.
.- *
*
.
I
SKELETAL
MUSCLE
-
ORIGIN
I
t
ORIGIN
+
+
Figure 1
Isoeoizymes separcated by starcch-gel elceirophoresis. A shows tile lactic deehydrogenases fromt
young rat tissues, separated and stained accordling to the method of Fine et al.2 B shows the
creatine kinases from the same tissues separated mtild stairoecl occeording to the nmethods of EPppenberger et al.25
Circulaton, Volumie XXXII, Decelmber 1965
1013
ISOENZYMES IN CLINICAL DIAGNOSIS
diversity of LDH isoenzymes in some individuals, resulting in more than five forms of this
enzyme.29 These appear to be mutations affecting the gene for one of the two subunit
types.
100
Physiologic Significance of Isoenzymes
80
60
20
x
;.e
40
20
oL
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33
10 4
3.3 lo0 3
PYRUVATE CONCENTRATION, M
10-3
Figure 2
Inhibition of two chicken isoenzymes of lactic dehydrogenase by pyruvate. The lines A and B indicate
two concentrations of pyruvate which could be used
to determine the relative amounts of the two isoenzymes (or their subunits) in a single sample,
(from Cahn et al.).23
subunits, analogous to the component chains
of hemoglobin or gamma globulin. A single
gene accounts for the structure of a single
subunit, but the intact enzyme can be composed of more than one subunit, and molecular
hybrids can occur. Thus, the five common isoenzymes of LDH are the products of only two
different genes, producing two kinds of subunits, which can combine in five different
ways to produce intact tetrameric enzyme
molecules.23 28 This is illustrated diagrammatically in figure 3. The gene for heart-type LDH
produces one kind of subunit, the H subunit,
and the gene for muscle-type LDH produces
M subunits. The complete enzyme contains
four subunits. In heart, the predominant tetramer is H4, in skeletal muscle it is M4, and,
in most other tissues, molecular hybrids of H
and M predominate. Thus, five isoenzymes
result from only two different genes. Enzymes that are dimers could have three isoenzymes as the result of two different genes
(fig. LB). The cell can thereby expand the
genetic complement into a wide spectrum of
isoenzyme patterns.
Genetic processes have given rise to further
Circulation, Volume XXXII, December 1965
The possible physiologic significance of isoenzymes is illustrated by lactic dehydrogenase. This enzyme catalyzes a reaction which,
in the direction pyruvate -> lactate, enables
glycolysis to provide energy in the absence
of oxygen. This reaction is important in tissues
such as skeletal and uterine muscle when energy from glycolysis is required during times of
reduced oxygenation. The isoenzymes of LDH
in these tissues are rich in muscle-type (M)
subunits, which have the property of functioning at high concentrations of pyruvate (fig.
2). Thus, these tissues can utilize the above
reaction when pyruvate cannot be oxidized.
On the other hand, the isoenzymes of LDH
found in heart are rich in H subunits and are
inhibited by high concentrations of pyruvate. This inhibition may retard the reaction
pyruvate -- lactate and promote the shunting
of pyruvate to oxidative pathways of the Krebs
cycle. In this way, the LDH isoenzymes in
heart muscle favor more complete utilization
of available energy in glucose. Furthermore,
the heart isoenzymes are better suited to
oxidize lactate; this may permit the heart to
extract lactate from the arterial blood and
use it as a metabolic fuel in addition to glu-
cose.23' 30
The other tissues of the body make intermediate demands on glycolysis and oxidation,
and their LDH is of intermediate type, con-
FM
SUBUNITS
TETRAMER
(ISOENZYME)
i 0i
i-m 9 i-m 0 i 0 0
W ( ((®®)
i®
i
(M4)
(M3H1) (M2H2) (M1H3)
im
i
®
®
(H4)
Figure 3
Diagrammatic representation of the formation of five
diferent isoenzymes from only two diferent subunits
(LDH), when the intact enzyme contains a total of
four subunits.
1014
GOODFRIEND, KAPLAN
tive and has not proved adequate by itself
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taining molecular hybrids of M and H subunits, and reacting in an intermediate way
with pyruvate. The fine adjustment of metabolism, which can be aided by various proportions of H and M subunits in LDH, is illustrated in table 3. The two zones of the kidney
vary in oxygen tension because of the countercurrent circulatory system: the medulla is anaerobic relative to the cortex.31 They also vary
in their dependence on anaerobic glycolysis
for energy: the medulla utilizes anaerobic
pathways more extensively than does the cortex.32 Finally, there is a parallel variation in the
proportion of subunits in the LDH from these
zones: the anaerobic medulla contains muscle
type, and the oxidative cortex contains mostly
heart-type enzyme.30 This difference in metabolic and isoenzyme pattern from cortex to
medulla may prove useful in the diagnosis and
localization of renal disease by tests on blood
or urine.
In addition to the differences observed in
tissues of adult organisms, the proportions of
the two kinds of LDH subunits can be made
to vary further by changes in environment.
In the uterus. estradiol induces preferential
synthesis of M subunits ("preparing" the organ
for its anaerobic, muscle-like adult function
In tissue culture,
during parturition) .
changes in oxygen tension alter the synthesis
of the subunits to different degrees.34 As might
be predicted, the synthesis of M subunits is
favored by anaerobic conditions. These observations reenforce the concept that isoenzyme differences serve a physiologic pur-
for the diagnosis of cancer.35
Differences in isoenzyme properties which
might correlate with physiology have also been
described for malic dehydrogenase, glutamic
dehydrogenase, and phosphofructokinase. The
malic dehydrogenase isoenzymes differ in their
resistance to high concentrations of malate,
depending on their subcellular site of origin,
that from mitochondria resisting high malate
better than that from the cytoplasm.36 3" This
is consistent with the fact that malate is oxidized primarily inside mitochondria. Glutamic
dehydrogenase isoenzymes vary in catalytic
activity. The less active form, which can be
produced in vitro by high concentrations of
ATP, probably predominates when oxidation
of glutamate for energy is unnecessary and
ATP levels are high. Oxidation of glutamate
by the more active form of enzyme is favored
when
energy
is needed and ATP is low.38
Finally,
phosphofructokinase from heart
is more resistant to ATP inhibition than the
enzyme from other tissues.30 This correlates
with the high levels of ATP in heart compared
to other tissues.
The isoenzymes of glutamic dehydrogenase,
which differ in molecular weight as well as
catalytic properties, are composed of varying
numbers of subunits. Furthermore, the isoenzymes are interconvertible, and the conversion appears to be under metabolic and endocrine control.40 This may represent the use of
isoenzymes by the cell for a constant fine adjustment of enzyme activity. Although the
majority of isoenzymes are not so readily interconvertible, many more examples of interconvertible forms will probably come to light.
If such interconversions were to occur among
pose.30
It has been shown that the proportion of M
subunits in tumors is higher than in normal
tissues. However, the difference is only rela-
Table 3
Comparison of Oxygen Supply, Oxygen Utilization, and Glycolysis with Composition
of LDH Isoenzymes in Zones of the Kidney from Various Species
Medulla
Cortex
(human) (mm. Hg),
(31)
Oxygen tension;
Glycolysis;
(dogs), (Al.
C02 released
from medium/
mg. dry wt.
hr.), (32)
20-60
90
5.1
1.4
ut(lg
zation; (dogs),
Oxygen
(-Qo,), (32)
M-subunits in
LDH: (rat), per
cent, (30)
2.3
14.1
56
2
Circulafion, Volume
XXXII, December 1965
ISOENZYMES IN CLINICAL DIAGNOSIS
1015
multiple forms, all of which were fairly stable,
the resulting isoenzyme patterns would provide accurate reflections of the momentary
physiologic (or pathologic) states in tissues.
ables that are subject to analysis. In the absence of absolutely organ-specific enzymes or
isoenzymes, the greater number of proteins
improves the diagnostic potential of this type
of test.
It may become possible to incorporate tests
for many relatively specific "iso-" proteins into
one over-all test by the use of antisera. Since
a classical means of identifying isoenzymes is
immunologic, it should be possible to develop
a series of organ-specific or tissue-specific antisera. If it were possible to detect the very
small antigen-antibody reactions that would
result when pathologic samples were exposed
to organ-specific antisera, this kind of test
would serve as a simultaneous screening procedure for a sum of many immunologically
distinct isoenzymes and other antigens.
Difficulties in Interpreting Isoenzyme
Patterns
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All cells share the same genes and are theoretically capable of producing the same proteins, including isoenzymes. Although there
are obvious marked differences in the localization of some proteins from tissue to tissue,
the differences in enzymes and isoenzymes are
likely to be relative rather than absolute. This
is the feature that has long obscured interpretation of standard serum enzyme tests. Use
of isoenzymes decreases this confusion somewhat, because it increases the number of vari-
Table 4
Selected List of Mammalian Enzymes with MIultiple Molecular Forms (Isoenzymes)
Enzyme
Usual number of isoenzymes
Lactic dehydrogenase
Creatine kinase
5
Phosphofructokinase
Aldolase
Phosphorylase
Aminopeptidase
Fructose 1, 6 diphosphatase
Ribonuclease
Hexokinase
Phosphatase
>2
Esterase
Deoxyribonuclease
Amylase
Glutamic dehydrogenase
Pepsin
Isocitric dehydrogenase
(TPN)
Malic dehydrogenase
Glutamic-oxalacetic
3
2
3
Isoenzymes
differ in
tissue or
organ localization
+
+
+
I soenzymes
differ in
catalytic
properties
or stability
+
+
Subunits
proved
cated
or indi-
+
+
+
+
>3
2
2
4
>3
>3
2
>2
6
4
+
+
+
+
+
+
(+)
+
Reference
no.
51,
54,
39
56
20,
14,
52, 53*
55*
57
58,* 59*
60
0
10, 61, 62*
63, 64, 65
2, 6, 16, 66,* 67*
13
68, 69*
70
38, 71
17, 72*
27
26, 37
2
>2
2
+
19, 73
2
+
74
>3
2
2
2
+
76
77
78
transaminase
Histidine-pyruvic transaminase
Glucose-6-phosphate
dehydrogenase
Galactokinase
Carbonic anhydrase
Tyrosinase
*Reports directly relat :ed to clinical medicine.
Circulation, Volume XXXII, December 1965
19, 75*
1016
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The preceding section described changes in
isoenzyme patterns that may be caused by
many physiologic or environmental conditions
in vivo. Recent evidence also indicates that
changes may occur depending on the time of
day, in accordance with a "biological clock"
mechanism.41 Furthermore, because they are
different molecules, isoenzymes are handled
differently by the body after they are released
into the circulation. The various transaminases
are cleared from the circulation at different
rates,42 and the clearance of lactic dehydrogenase in mice is affected by a virus.43 As
mentioned in the first section of this review,
many changes in isoenzyme pattern can be
induced by heat, acid, and solvents. Finally,
the isoenzymes that result from various combinations of subunits often can be dissociated
and reassociated in vitro.44 45 Because of this
recombination, a sample that has been frozen
and thawed may exhibit a different isoenzyme
pattern from the fresh sample.
Thus, physiologic, pathologic, and physicochemical factors bear upon the isoenzyme pattern during its passage through the cell, circulation, and laboratory. At the very least,
conditions of collection, storage, separation,
and assay must be standardized before isoenzyme comparisons are valid.
Summary
The recent literature attests to the increasing number of enzymes for which isoenzymes
or subunit structure or both are known. Several symposia and reviews have been published within the past few years.4>50 Some
enzyme activities for which isoenzymes have
been described are listed in table 4. Only
those found in mammals are included. They
are ranked in rough order of apparent usefulness in clinical diagnosis, with the enzymes
displaying tissue localization and catalytic differences listed first. Also, the number of molecular forms and the presence or absence of
known subunit structure is indicated.
Clinical studies have been made using several of the isoenzymes listed in table 4, and
references denoting these studies are marked
with an asterisk. Data presented in the table
GOODFRIEND, KAPLAN
indicate that valuable clinical information
might derive from study of other isoenzymes,
notably phosphofructokinase, for which tissue
differences and adaptable catalytic differences
exist. The guidelines set forth in this review
can be used for investigating still other isoenzymes, such as those studied in lower
forms.79-85 In brief, the study of isoenzymes,
like the study of enzymes and other organspecific chemicals, presents an opportunity for
great specificity and accuracy in localizing
and following disease processes.
Acknowledgment
The authors are indebted to Dr. H. Eppenberger
for figure 1, and to Dr. L. Corman and Mr. J. Everse
for assistance in translation.
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Circulation, Volume XXXII, December 1965
1017
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Isoenzymes in Clinical Diagnosis
THEODORE L. GOODFRIEND and NATHAN O. KAPLAN
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