SPECIAL ARTICLE

Isoenzymes in Clinical Diagnosis

By THEODORE L. GOODFRIEND, M.D., AND NATHAN 0. KAPLAN, PH.D.
SERUM ENZYMES are useful tools for de- The acid and alkaline phosphatases are not
termining the location and severity of usually designated as isoenzymes. This term
many diseases. They are protein catalysts, was first applied to enzymes that catalyzed
some of which enter the serum from damaged the same reaction but differed from one anoth-
tissues. Because they are catalysts, they are er in electrophoretic or chromatographic prop-
more easily detected than many other sub- erties. For the purposes of this review, the
stances. Amylase released from the diseased definition of isoenzymes will be broadened to
pancreas,1 and phosphatase released from include all sets of enzymes that catalyze a
several tissues2 have been studied for many given reaction, regardless of the nature of
years and have established the usefulness of the differences among them. By this definition,
serum enzyme tests. acid and alkaline phosphatases are isoenzymes,
Unfortunately, many of the best studied and the recently described electrophoretic
and most easily detected enzymes occur in forms within each of these groups are also
more than one organ. Furthermore, some or- isoenzymes.
gans like liver and skeletal muscle contain
Characteristics of Isoenzymes
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high concentrations of many enzymes and

frequently cause confusion in diagnosis Enzymes that catalyze the same reaction
based on enzymes. may differ from one another in many ways,
In one respect, however, the wide distribu- ranging from small variations in secondary
tion of some enzymes is illusory: the enzyme structure to broad differences in amino acid
activity is widespread, but the specific protein sequence and molecular weight. These criteria
catalyst may vary from tissue to tissue. This are listed in table 1. At one extreme are en-
was recognized early in the case of phos- zymes with marked differences in structure,
phatases.3 Bone, prostate, and red cells are but a common substrate. The esterases13 and
rich in proteins catalyzing the hydrolysis of the peptidases'4 are probably in this class. At
phosphates, but the major phosphatases from the other extreme are molecules that are iden-
these tissues differ in pH optima and suscepti- tical in all respects but their degree of dena-
bility to inhibition by a variety of chemicals. turation. Separatory procedures are now so
Different enzyme molecules that catalyze the sensitive that multiple forms of enzymes may
same reaction are called "isoenzymes," 'iso- appear, which merely reflect artifacts of prep-
zymes," or "multiple molecular forms." Their aration or handling of the specimens. Rela-
discovery has encouraged further searches for tively minor manipulations may introduce dif-
organ-specific catalytic proteins. ferences caused by the folding of enzyme
protein chains,4 the aggregation of chains into
From the Graduate Department of Biochemistry, polymers,5 the addition or removal of lipids,11
Brandeis University, Waltham, Massachusetts. Pub- bound metals,15 or deamidation of carboxyl
lication no. 393. amide groups.8 For clinical studies, the
Supported in part by grants from the National In-
stitutes of Health, U. S. Public Health Service, and isoenzymes with the greatest value are those
the National Science Foundation. that differ in a fundamental way, which per-
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

sists despite the influence of serum contami- small amounts of enzyme, and various condi-
nants, storage, or analytic technics. tions of heat treatment, pH, substrate con-
centration, or inhibitors. Such assays do not
Detection of Isoenzymes require preliminary separation of the pro-
Two groups of phosphatase were originally teins by chromatography, etc. The heart and
detected by their different pH optima and muscle forms of LDH are examples of iso-
called "acid" and "alkaline" phosphatase. Fur- enzymes detectable by such chemical proper-
ther "isoenzymes" of phosphatase were dif- ties. In fact, the demonstration of experimen-
ferentiated by susceptibility to inhibition by tal data like that shown in figure 2 is an
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tartrate and other chemicals.'6 Different pep- indication that such methods are applicable.
sins were separated by differences in solubil- These curves indicate at a glance the condi-
ity.17 Since these early examples, methods of tions of assay that will differentiate the two
differentiation have increased. The most wide- forms (shown by the vertical dotted lines),
ly used are technics of separation by electro- one condition measures both forms equally
phoresis or chromatography. The basic meth- well, the second condition specifically inhibits
ods of isoenzyme detection and measurement one of the two forms. Such an assay is pre-
are listed in table 2. cise and quantitative. If more than two iso-
Figure 1A is an example of lactic dehydro- enzymes are present, however, this type of
genase isoenzymes (LDH), separated by elec- test may not give as complete a picture of the
trophoresis and detected by a staining reaction spectrum of isoenzymes as electrophoresis or
specific for this enzyme. Figure lB shows an chromatography. Finally it may be pos-
analogous pattern for creatine kinase. Once sible to adapt separatory procedures to a sort
separated, the isoenzymes may prove to have of chemical assay. For example, isoenzymes
different catalytic properties. For example, the that adhere relatively firmly to materials used
muscle and heart varieties of LDH behave in chromatography can be removed from as-
differently toward various concentrations of say mixtures by the "batch" addition of gel to
their common substrate, as shown in figure the specimen. In this way, the gel is used
2. These differences, like different susceptibil- instead of a chemical inhibitor.
ity to pH, inhibitors, and specific antibodies,
may be utilized for detecting and measuring Origin of Isoenzymes
the various enzyme forms. The isoenzymes that differ in amino acid
The isoenzymes that are currently most con- composition, such as the isoenzymes of LDH,
venient for clinical diagnosis are those which probably represent the results of ancestral
can be differentiated by simple assays using mutation and gene duplication: a single gene
Circulation, Volume XXXII, December 1965
 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

corresponding to a giveni enzyme gave rise to proteins uvhich share a common ftinction, such
two or more genes and two or more different as hemoglobins, haptoglobins, and gamma
enzymes. Those isoenzymes that differ in globulins. Another exam-ple is the hormone-
prosthetic groups, secondary structture, or pair oxytocin aind vasopressin, xhich share
state of polymerization may have arisen by common properties and probably arose from
other routes. In some cases, further evolu- a single ancestor like the vasotocin of loNver
tion or environmental influences caused sepa- forms. It has lonig been recognized that some
rate genes to become expressed to varying ssuch "iso" proteins vary from species to spe-
degrees in different organs. This resuilted in the cies, from race to race, or from person to per-
organ-specific isoenizyme patterns under dis- son. The coexistence of mutltiple forms of en-
cussion. There are other instances, niotably zymes or other proteins within the same
malic.' and TPN-linked isocitric dehydrogen- individual but localized in various organs is
ases$2 in whiclh the isoenzymes are found in the featuire that makes them useful in clinical
many tissues l)ut are localized in different sub- diagnosis.
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cellular compartments, such as mitochondria The existence of two different genes for
anid cytoplasm. fuinctionally related enzymes may give rise to
Genetic and evolutionary factors havce more than tu0o isoenizymes. This resuilts from
given rise to differences in other well-studied the fact that some enzy mes are composed of
A S
LACTIC DEHYDROGENASE CREATINE KINASE
LDH
ISOENZYMES M4 M3}H M2H2 MMH3 H
MHMH2MHH4
BRAI N I
BRAIN
HEART
HEART
'Ro. .- * * . SKELETAL
SKELETAL MUSCLE I
I
MUSCLE
-
t
ORIGIN +
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 EPp-
penberger et al.25
Circulaton, Volumie XXXII, Decelmber 1965
 ISOENZYMES IN CLINICAL DIAGNOSIS 1013

diversity of LDH isoenzymes in some individ-

uals, resulting in more than five forms of this
enzyme.29 These appear to be mutations af-
fecting the gene for one of the two subunit
100
types.
80 Physiologic Significance of Isoenzymes
The possible physiologic significance of iso-
60 enzymes is illustrated by lactic dehydrogen-
x
20
ase. This enzyme catalyzes a reaction which,
;.e 40 in the direction pyruvate -> lactate, enables
glycolysis to provide energy in the absence
20 of oxygen. This reaction is important in tissues
such as skeletal and uterine muscle when ener-
oL
33 10 4 10-3 3.3 lo0 3
gy from glycolysis is required during times of
PYRUVATE CONCENTRATION, M reduced oxygenation. The isoenzymes of LDH
Figure 2 in these tissues are rich in muscle-type (M)
subunits, which have the property of function-
Inhibition of two chicken isoenzymes of lactic de- ing at high concentrations of pyruvate (fig.
hydrogenase by pyruvate. The lines A and B indicate
two concentrations of pyruvate which could be used 2). Thus, these tissues can utilize the above
to determine the relative amounts of the two iso- reaction when pyruvate cannot be oxidized.
enzymes (or their subunits) in a single sample, On the other hand, the isoenzymes of LDH
(from Cahn et al.).23 found in heart are rich in H subunits and are
inhibited by high concentrations of pyru-
subunits, analogous to the component chains vate. This inhibition may retard the reaction
of hemoglobin or gamma globulin. A single
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pyruvate -- lactate and promote the shunting

gene accounts for the structure of a single
of pyruvate to oxidative pathways of the Krebs
subunit, but the intact enzyme can be com- cycle. In this way, the LDH isoenzymes in
posed of more than one subunit, and molecular heart muscle favor more complete utilization
hybrids can occur. Thus, the five common iso- of available energy in glucose. Furthermore,
enzymes of LDH are the products of only two
the heart isoenzymes are better suited to
different genes, producing two kinds of sub- oxidize lactate; this may permit the heart to
units, which can combine in five different extract lactate from the arterial blood and
ways to produce intact tetrameric enzyme use it as a metabolic fuel in addition to glu-
molecules.23 28 This is illustrated diagrammati- cose.23' 30
cally in figure 3. The gene for heart-type LDH The other tissues of the body make inter-
produces one kind of subunit, the H subunit, mediate demands on glycolysis and oxidation,
and the gene for muscle-type LDH produces and their LDH is of intermediate type, con-
M subunits. The complete enzyme contains
four subunits. In heart, the predominant te-
tramer is H4, in skeletal muscle it is M4, and,
SUBUNITS FM
in most other tissues, molecular hybrids of H
and M predominate. Thus, five isoenzymes TETRAMER im i i-m 9 i-m 0 i 0 0
result from only two different genes. En- (ISOENZYME) i 0i i® i W ( ((®®) ® ®

zymes that are dimers could have three iso- (M4) (M3H1) (M2H2) (M1H3) (H4)
enzymes as the result of two different genes
Figure 3
(fig. LB). The cell can thereby expand the Diagrammatic representation of the formation of five
genetic complement into a wide spectrum of diferent isoenzymes from only two diferent subunits
isoenzyme patterns. (LDH), when the intact enzyme contains a total of
Genetic processes have given rise to further four subunits.
Circulation, Volume XXXII, December 1965
 1014 GOODFRIEND, KAPLAN

taining molecular hybrids of M and H sub- tive and has not proved adequate by itself
units, and reacting in an intermediate way for the diagnosis of cancer.35
with pyruvate. The fine adjustment of metab- Differences in isoenzyme properties which
olism, which can be aided by various propor- might correlate with physiology have also been
tions of H and M subunits in LDH, is illus- described for malic dehydrogenase, glutamic
trated in table 3. The two zones of the kidney dehydrogenase, and phosphofructokinase. The
vary in oxygen tension because of the counter- malic dehydrogenase isoenzymes differ in their
current circulatory system: the medulla is an- resistance to high concentrations of malate,
aerobic relative to the cortex.31 They also vary depending on their subcellular site of origin,
in their dependence on anaerobic glycolysis that from mitochondria resisting high malate
for energy: the medulla utilizes anaerobic better than that from the cytoplasm.36 3" This
pathways more extensively than does the cor- is consistent with the fact that malate is oxi-
tex.32 Finally, there is a parallel variation in the dized primarily inside mitochondria. Glutamic
proportion of subunits in the LDH from these dehydrogenase isoenzymes vary in catalytic
zones: the anaerobic medulla contains muscle activity. The less active form, which can be
type, and the oxidative cortex contains mostly produced in vitro by high concentrations of
heart-type enzyme.30 This difference in meta- ATP, probably predominates when oxidation
bolic and isoenzyme pattern from cortex to of glutamate for energy is unnecessary and
medulla may prove useful in the diagnosis and ATP levels are high. Oxidation of glutamate
localization of renal disease by tests on blood by the more active form of enzyme is favored
or urine. when energy is needed and ATP is low.38
In addition to the differences observed in Finally, phosphofructokinase from heart
tissues of adult organisms, the proportions of is more resistant to ATP inhibition than the
the two kinds of LDH subunits can be made enzyme from other tissues.30 This correlates
to vary further by changes in environment. with the high levels of ATP in heart compared
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In the uterus. estradiol induces preferential to other tissues.

synthesis of M subunits ("preparing" the organ The isoenzymes of glutamic dehydrogenase,
for its anaerobic, muscle-like adult function which differ in molecular weight as well as
during parturition) . In tissue culture, catalytic properties, are composed of varying
changes in oxygen tension alter the synthesis numbers of subunits. Furthermore, the iso-
of the subunits to different degrees.34 As might enzymes are interconvertible, and the conver-
be predicted, the synthesis of M subunits is sion appears to be under metabolic and endo-
favored by anaerobic conditions. These ob- crine control.40 This may represent the use of
servations reenforce the concept that isoen- isoenzymes by the cell for a constant fine ad-
zyme differences serve a physiologic pur- justment of enzyme activity. Although the
pose.30 majority of isoenzymes are not so readily in-
It has been shown that the proportion of M terconvertible, many more examples of inter-
subunits in tumors is higher than in normal convertible forms will probably come to light.
tissues. However, the difference is only rela- If such interconversions were to occur among
Table 3
Comparison of Oxygen Supply, Oxygen Utilization, and Glycolysis with Composition
of LDH Isoenzymes in Zones of the Kidney from Various Species
Glycolysis;
(dogs), (Al.
C02 released
Oxygen tension; from medium/ Oxygen ut(lg M-subunits in
(human) (mm. Hg), mg. dry wt. zation; (dogs), LDH: (rat), per
(31) hr.), (32) (-Qo,), (32) cent, (30)
Medulla 20-60 5.1 2.3 56
Cortex 90 1.4 14.1 2

Circulafion, Volume XXXII, December 1965

 ISOENZYMES IN CLINICAL DIAGNOSIS 1015
multiple forms, all of which were fairly stable, ables that are subject to analysis. In the ab-
the resulting isoenzyme patterns would pro- sence of absolutely organ-specific enzymes or
vide accurate reflections of the momentary isoenzymes, the greater number of proteins
physiologic (or pathologic) states in tissues. improves the diagnostic potential of this type
of test.
Difficulties in Interpreting Isoenzyme It may become possible to incorporate tests
Patterns for many relatively specific "iso-" proteins into
All cells share the same genes and are the- one over-all test by the use of antisera. Since
oretically capable of producing the same pro- a classical means of identifying isoenzymes is
teins, including isoenzymes. Although there immunologic, it should be possible to develop
are obvious marked differences in the localiza- a series of organ-specific or tissue-specific an-
tion of some proteins from tissue to tissue, tisera. If it were possible to detect the very
the differences in enzymes and isoenzymes are small antigen-antibody reactions that would
likely to be relative rather than absolute. This result when pathologic samples were exposed
is the feature that has long obscured inter- to organ-specific antisera, this kind of test
pretation of standard serum enzyme tests. Use would serve as a simultaneous screening pro-
of isoenzymes decreases this confusion some- cedure for a sum of many immunologically
what, because it increases the number of vari- distinct isoenzymes and other antigens.
Table 4
Selected List of Mammalian Enzymes with MIultiple Molecular Forms (Isoenzymes)
Isoenzymes I soenzymes
differ in differ in Subunits
Usual num- tissue or catalytic proved
ber of iso- organ lo- properties or indi- Reference
Enzyme enzymes calization or stability cated no.
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Lactic dehydrogenase 5 51, 52, 53*

Creatine kinase 3 + + + 54, 55*
Phosphofructokinase >2 + + 39
Aldolase 2 + + + 56
Phosphorylase 3 + 20, 57
Aminopeptidase >3 14, 58,* 59*
Fructose 1, 6 diphos- + +
phatase 2 + 60
Ribonuclease 2 + + 0 10, 61, 62*
Hexokinase 4 63, 64, 65
Phosphatase >3 2, 6, 16, 66,* 67*
Esterase >3 + 13
Deoxyribonuclease 2 (+) 68, 69*
Amylase >2 + 70
Glutamic dehydrogenase 6 38, 71
Pepsin 4 17, 72*
Isocitric dehydrogenase
(TPN) 2 27
Malic dehydrogenase >2 26, 37
Glutamic-oxalacetic
transaminase 2 + 19, 73
Histidine-pyruvic trans-
aminase 2 + 74
Glucose-6-phosphate
dehydrogenase >3 19, 75*
Galactokinase 2 + 76
Carbonic anhydrase 2 77
Tyrosinase 2 78
*Reports directly relat :ed to clinical medicine.
Circulation, Volume XXXII, December 1965
 1016 GOODFRIEND, KAPLAN
The preceding section described changes in indicate that valuable clinical information
isoenzyme patterns that may be caused by might derive from study of other isoenzymes,
many physiologic or environmental conditions notably phosphofructokinase, for which tissue
in vivo. Recent evidence also indicates that differences and adaptable catalytic differences
changes may occur depending on the time of exist. The guidelines set forth in this review
day, in accordance with a "biological clock" can be used for investigating still other iso-
mechanism.41 Furthermore, because they are enzymes, such as those studied in lower
different molecules, isoenzymes are handled forms.79-85 In brief, the study of isoenzymes,
differently by the body after they are released
into the circulation. The various transaminases like the study of enzymes and other organ-
are cleared from the circulation at different specific chemicals, presents an opportunity for
rates,42 and the clearance of lactic dehydro- great specificity and accuracy in localizing
genase in mice is affected by a virus.43 As and following disease processes.
mentioned in the first section of this review,
many changes in isoenzyme pattern can be Acknowledgment
induced by heat, acid, and solvents. Finally, The authors are indebted to Dr. H. Eppenberger
for figure 1, and to Dr. L. Corman and Mr. J. Everse
the isoenzymes that result from various com- for assistance in translation.
binations of subunits often can be dissociated
and reassociated in vitro.44 45 Because of this References
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 ISOENZYMES IN CLINICAL DIAGNOSIS 1017
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