13

Enzymes
C H A P T E R
KAMISHA L. JOHNSON-DAVIS

CHAPTER OUTLINE

u GENERAL PROPERTIES AND DEFINITIONS Lactate Dehydrogenase

u ENZYME CLASSIFICATION AND Aspartate Aminotransferase
NOMENCLATURE Alanine Aminotransferase
u ENZYME KINETICS Alkaline Phosphatase
Catalytic Mechanism of Enzymes Acid Phosphatase
Factors That Influence Enzymatic γ-Glutamyltransferase
Reactions Amylase
Measurement of Enzyme Activity Lipase
Calculation of Enzyme Activity Glucose-6-Phosphate Dehydrogenase
Measurement of Enzyme Mass Drug-Metabolizing Enzymes
Enzymes as Reagents u QUESTIONS
u ENZYMES OF CLINICAL SIGNIFICANCE u REFERENCES
Creatine Kinase

Chapter Objectives various disorders, including cardiac, hepatic, bone, and

Upon completion of this chapter, the clinical muscle, malignancies, and acute pancreatitis.
laboratorian should be able to do the following: Discuss the tissue sources, diagnostic significance, and
assays, including sources of error, for the following
Define the term enzyme, including physical composition enzymes: creatine kinase, lactate dehydrogenase, aspar-
and structure. tate aminotransferase, alanine aminotransferase, alkaline
Classify enzymes according to the International Union of phosphatase, acid phosphatase, γ-glutamyltransferase,
Biochemistry. amylase, lipase, cholinesterase, and glucose-6-phosphate
Discuss the different factors affecting the rate of an dehydrogenase.
enzymatic reaction. Evaluate patient serum enzyme levels in relation to disease
Explain enzyme kinetics including zero-order and states.
first-order kinetics. Discuss the clinical importance for detecting
Explain why the measurement of serum enzyme levels is macroenzymes.
clinically useful. Discuss the role of enzymes in drug metabolism.
Discuss which enzymes are useful in the diagnosis of

KEY TERMS Hydrolase

International unit (IU)
Activation energy Isoenzyme
Activators Isoform
Apoenzyme Kinetic assay
Coenzyme LD flipped pattern
Cofactor Michaelis-Menten constant
Enzyme Oxidoreductase
Enzyme–substrate (ES) complex Transferase
First-order kinetics Zero-order kinetics
Holoenzyme Zymogen

262
 CHAPTER 13 n ENZYMES 263

Enzymes are specific biologic proteins that catalyze dinucleotide (NAD). When bound tightly to the enzyme,
biochemical reactions without altering the equilibrium the coenzyme is called a prosthetic group. The enzyme
point of the reaction or being consumed or changed portion (apoenzyme), with its respective coenzyme,
in composition. The other substances in the reaction forms a complete and active system, a holoenzyme.
are converted to products. The catalyzed reactions are Some enzymes, mostly digestive enzymes, are origi-
frequently specific and essential to physiologic func- nally secreted from the organ of production in a struc-
tions, such as the hydration of carbon dioxide, nerve turally inactive form, called a proenzyme or zymogen.
conduction, muscle contraction, nutrient degradation, Other enzymes later alter the structure of the proenzyme
and energy use. Found in all body tissues, enzymes to make active sites available by hydrolyzing specific
frequently appear in the serum following cellular injury amino acid residues. This mechanism prevents digestive
or, sometimes, in smaller amounts, from degraded cells. enzymes from digesting their place of synthesis.
Certain enzymes, such as those that facilitate coagula-
tion, are specific to plasma and, therefore, are pres- ENZYME CLASSIFICATION AND
ent in significant concentrations in plasma. Plasma or NOMENCLATURE
serum enzyme levels are often useful in the diagnosis
To standardize enzyme nomenclature, the Enzyme
of particular diseases or physiologic abnormalities. This
Commission (EC) of the IUB adopted a classification
chapter discusses the general properties and principles
system in 1961; the standards were revised in 1972 and
of enzymes, aspects relating to the clinical diagnostic
1978. The IUB system assigns a systematic name to each
significance of specific physiologic enzymes, and assay
enzyme, defining the substrate acted on, the reaction
methods for those enzymes.
catalyzed, and, possibly, the name of any coenzyme
involved in the reaction. Because many systematic names
GENERAL PROPERTIES AND DEFINITIONS
are lengthy, a more usable, trivial, recommended name is
Enzymes catalyze many specific physiologic reactions. also assigned by the IUB system.1
These reactions are facilitated by the enzyme structure In addition to naming enzymes, the IUB system iden-
and several other factors. As a protein, each enzyme con- tifies each enzyme by an EC numerical code containing
tains a specific amino acid sequence (primary structure), four digits separated by decimal points. The first digit
-

with the resultant polypeptide chains twisting (second- places the enzyme in one of the following six classes:
ary structure), which then folds (tertiary structure) and
1. Oxidoreductases. Catalyze an oxidation–reduction
results in structural cavities. If an enzyme contains more
reaction between two substrates
than one polypeptide unit, the quaternary structure refers
2. Transferases. Catalyze the transfer of a group other
to the spatial relationships between the subunits. Each
than hydrogen from one substrate to another
enzyme contains an active site, often a water-free cav-
3. Hydrolases. Catalyze hydrolysis of various bonds
ity, where the substance on which the enzyme acts (the
4. Lyases. Catalyze removal of groups from substrates
substrate) interacts with particular charged amino acid
without hydrolysis; the product contains double
residues. An allosteric site—a cavity other than the active
bonds
site—may bind regulator molecules and, thereby, be sig-
5. Isomerases. Catalyze the interconversion of geomet-
nificant to the basic enzyme structure.
ric, optical, or positional isomers
Even though a particular enzyme maintains the same
6. Ligases. Catalyze the joining of two substrate mol-
catalytic function throughout the body, that enzyme may
ecules, coupled with breaking of the pyrophosphate
exist in different forms within the same individual. The
bond in adenosine triphosphate (ATP) or a similar
different forms may be differentiated from each other
compound
based on certain physical properties, such as electropho-
retic mobility, solubility, or resistance to inactivation. The second and third digits of the EC code number
The term isoenzyme is generally used when discuss- represent the subclass and subsubclass of the enzyme,
ing such enzymes; however, the International Union of respectively, divisions that are made according to criteria
Biochemistry (IUB) suggests restricting this term to mul- specific to the enzymes in the class. The final number is
tiple forms of genetic origin. An isoform results when the serial number specific to each enzyme in a subsub-
an enzyme is subject to posttranslational modifications. class. Table 13-1 provides the EC code numbers, as well
Isoenzymes and isoforms contribute to heterogeneity in as the systematic and recommended names, for enzymes
properties and function of enzymes. frequently measured in the clinical laboratory.
In addition to the basic enzyme structure, a nonpro- Table 13-1 also lists common and standard abbre-
tein molecule, called a cofactor, may be necessary for viations for commonly analyzed enzymes. Without IUB
enzyme activity. Inorganic cofactors, such as chloride recommendation, capital letters have been used as a
or magnesium ions, are called activators. A coenzyme convenience to identify enzymes. The common abbre-
is an organic cofactor, such as nicotinamide adenine viations, sometimes developed from previously accepted
264 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

TABLE 13-1 CLASSIFICATION OF FREQUENTLY QUANTITATED ENZYMES

RECOMMENDED COMMON STANDARD EC CODE
CLASS NAME ABBREVIATION ABBREVIATION NO. SYSTEMATIC NAME
Oxidoreductases Lactate LD LD 1.1.1.27 L-Lactate:NAD+
dehydrogenase oxidoreductase
Glucose-6-phosphate G-6-PDH G-6-PD 1.1.1.49 D-Glucose-6-
dehydrogenase phosphate:NADP+
1-oxidoreductase
Glutamate GLD GLD 1.4.1.3 L-glutamate:NAD(P)
dehydrogenase oxidoreductase,
deaminase
Transferases Aspartate GOT (glutamate AST 2.6.1.1 L-Aspartate:2-
aminotransferase oxaloacetate oxaglutarate
transaminase) aminotransferase
Alanine GPT (glutamate ALT 2.6.1.2 L-Alanine:2-oxaglutarate
aminotransferase transaminase) aminotransferase
Creatine kinase CPK (creatine CK 2.7.3.2 ATP:creatine
phosphokinase) N-phosphotransferase
γ-Glutamyltransferase GGTP GGT 2.3.2.2 (5-Glutamyl)peptide:
amino acid-5-glutamyl-
transferase
Glutathione-S- α-GST GST 2.5.1.18 Glutathione transferase
transferase
Glycogen GP GP 2.4.1.1 1,4-D-Glucan:
phosphorylase orthophosphate α-D-
glucosyltransferase
Pyruvate kinase PK PK 2.7.1.40 Pyruvate kinase
Hydrolases Alkaline ALP ALP 3.1.3.1 Orthophosphoric
phosphatase monoester phospho-
hydrolase (alkaline
optimum)
Acid phosphatase ACP ACP 3.1.3.2 Orthophosphoric
monoester phosphohy-
drolase (acid optimum)
α-Amylase AMS AMY 3.2.1.1 1,4-D-Glucan
glucanohydrolase
Cholinesterase PCHE CHE 3.1.1.8 Acylcholine acylhydrolase
Chymotrypsin CHY CHY 3.4.21.1 Chymotrypsin
Elastase-1 E1 E1 3.4.21.36 Elastase
5-Nucleotidase NTP NTP 3.1.3.5 5′-Ribonucleotide
phosphohydrolase
Triacylglycerol lipase LPS 3.1.1.3 Triacylglycerol
acylhydrolase
Trypsin TRY TRY 3.4.21.4 Trypsin
Lyases Aldolase ALD ALD 4.1.2.13 D-D-Fructose-1,6-bisdi-
phosphateD-glyceral-
dehyde-3-phosphate-
lyase
Isomerases Triosephosphate TPI TPI 5.3.1.1 Triose-phosphate
isomerase isomerase
Ligase Glutathione GSH-S GSH-S 6.3.2.3 Glutathione synthase
synthetase
Adapted from Competence Assurance, ASMT. Enzymology, an Educational Program. Bethesda, MD: RMI Corporation; 1980.
 CHAPTER 13 n ENZYMES 265

names for the enzymes, were used until the standard The general relationship among the enzyme, substrate,
abbreviations listed in the table were developed.2,3 These and product may be represented as follows:
standard abbreviations are used in the United States and
E + S → ES → E + P (Eq. 13-1)
are used later in this chapter to indicate specific enzymes.
where E is the enzyme, S is the substrate, ES is the
ENZYME KINETICS enzyme–substrate complex, and P is the product.
The ES complex is a physical binding of a substrate
Catalytic Mechanism of Enzymes to the active site of an enzyme. The structural arrange-
A chemical reaction may occur spontaneously if the ment of amino acid residues within the enzyme makes
Ksp free energy or available kinetic energy is higher for the the three-dimensional active site available. At times, the
reactants than for the products. The reaction then pro- binding of ligand drives active site rearrangement. The
ceeds toward the lower energy if a sufficient number of transition state for the ES complex has a lower energy of
the reactant molecules possess enough excess energy activation than the transition state of S alone, so that the
to break their chemical bonds and collide to form new reaction proceeds after the complex is formed. An actual
bonds. The excess energy, called activation energy, is reaction may involve several substrates and products.
the energy required to raise all molecules in 1 mol of Different enzymes are specific to substrates in differ-
a compound at a certain temperature to the transition ent extents or respects. Certain enzymes exhibit absolute
state at the peak of the energy barrier. At the transition specificity, meaning that the enzyme combines with only
state, each molecule is equally likely to either participate one substrate and catalyzes only the one correspond-
in product formation or remain an unreacted molecule. ing reaction. Other enzymes are group specific because
Reactants possessing enough energy to overcome the they combine with all substrates containing a particular
energy barrier participate in product formation. chemical group, such as a phosphate ester. Still other
One way to provide more energy for a reaction is enzymes are specific to chemical bonds and thereby
to increase the temperature and thus increase intermo- exhibit bond specificity.
lecular collisions; however, this does not normally occur Stereoisometric specificity refers to enzymes that pre-
physiologically. Enzymes catalyze physiologic reactions dominantly combine with only one optical isomer of
by lowering the activation energy level that the reac- a certain compound. In addition, an enzyme may bind
tants (substrates) must reach for the reaction to occur more than one molecule of substrate, and this may
(Fig. 13-1). The reaction may then occur more readily to occur in a cooperative fashion. Binding of one substrate
a state of equilibrium in which there is no net forward or molecule, therefore, may facilitate binding of additional
reverse reaction, even though the equilibrium constant substrate molecules.
of the reaction is not altered. The extent to which the
reaction progresses depends on the number of substrate Factors That Influence Enzymatic Reactions
molecules that pass the energy barrier. Substrate Concentration
The rate at which an enzymatic reaction proceeds and
whether the forward or reverse reaction occurs depend
on several reaction conditions. One major influence
on enzymatic reactions is substrate concentration. In
1913, Michaelis and Menten hypothesized the role
of substrate concentration in the formation of the
enzyme–substrate (ES) complex. According to their
hypothesis, represented in Figure 13-2, the substrate
readily binds to free enzyme at a low substrate con-
centration. With the amount of enzyme exceeding the
amount of substrate, the reaction rate steadily increases
as more substrate is added. The reaction is following
first-order kinetics because the reaction rate is directly
proportional to substrate concentration. Eventually,
however, the substrate concentration is high enough to
saturate all available enzyme, and the reaction velocity
reaches its maximum. When the product is formed,
the resultant free enzyme immediately combines with
FIGURE 13-1  Energy vs. progression of reaction, indicating the
energy barrier that the substrate must surpass to react with and
excess free substrate. The reaction is in zero-order
without enzyme catalysis. The enzyme considerably reduces the kinetics, and the reaction rate depends only on enzyme
free energy needed to activate the reaction. concentration.
266 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

Vmax
Km

FIGURE 13-2 Michaelis-Menten curve of velocity vs. substrate con- Km

centration for enzymatic reaction. Km is the substrate concentration FIGURE 13-3 Lineweaver-Burk transformation of Michaelis-Menten
at which the reaction velocity is half of the maximum level. curve. Vmax is the reciprocal of the x-intercept of the straight line.
Km is the negative reciprocal of the x-intercept of the same line.

The Michaelis-Menten constant (Km), derived from

the theory of Michaelis and Menten, is a constant for
a specific enzyme and substrate under defined reac- taken of both the substrate concentration and the velocity
tion conditions and is an expression of the relationship of an enzymatic reaction. The equation becomes
between the velocity of an enzymatic reaction and sub- 1 _____ K
1
m ___ 1
__ = + _____ (Eq. 13-3)
strate concentration. The assumptions are made that V Vmax [S] Vmax
equilibrium among E, S, ES, and P is established rapidly
and that the E + P → ES reaction is negligible. The rate- Enzyme Concentration
limiting step is the formation of product and enzyme Because enzymes catalyze physiologic reactions, the
from the ES complex. Then, maximum velocity is fixed, enzyme concentration affects the rate of the catalyzed
and the reaction rate is a function of only the enzyme reaction. As long as the substrate concentration exceeds
concentration. As designated in Figure 13-2, Km is spe- the enzyme concentration, the velocity of the reaction is
cifically the substrate concentration at which the enzyme proportional to the enzyme concentration. The higher
yields half the possible maximum velocity. Therefore, Km the enzyme level, the faster the reaction will proceed
indicates the amount of substrate needed for a particular because more enzyme is present to bind with the sub-
enzymatic reaction. strate.
The Michaelis-Menten hypothesis of the relationship
between reaction velocity and substrate concentration pH
can be represented mathematically as follows: Enzymes are proteins that carry net molecular charges.
Vmax[S] Changes in pH may denature an enzyme or influence its
V = ________ (Eq. 13-2) ionic state, resulting in structural changes or a change
Km + [S] in the charge of an amino acid residue in the active site.
Hence, each enzyme operates within a specific pH range
where V is the measured velocity of reaction, Vmax is the
and maximally at a specific pH. Most physiologic enzy-
maximum velocity, [S] is the substrate concentration,
matic reactions occur in the pH range of 7.0 to 8.0, but
and Km is the Michaelis-Menten constant of enzyme for
some enzymes are active in wider pH ranges than others.
a specific substrate.
In the laboratory, the pH for a reaction is carefully con-
Theoretically, Vmax and then Km could be determined
trolled at the optimal pH by means of appropriate buffer
from the plot in Figure 13-2. However, Vmax is difficult to
solutions.
determine from the hyperbolic plot and often not actually
achieved in enzymatic reactions because enzymes may not Temperature
function optimally in the presence of excessive substrate. Increasing the temperature usually increases the rate
A more accurate and convenient determination of Vmax of a chemical reaction by increasing the movement of
and Km may be made through a Lineweaver-Burk plot, a molecules, the rate at which intermolecular collisions
double-reciprocal plot of the Michaelis-Menten constant, occur, and the energy available for the reaction. This is
which yields a straight line (Fig. 13-3). The reciprocal is the case with enzymatic reactions until the temperature
 CHAPTER 13 n ENZYMES 267

is high enough to denature the protein composition of Inhibitors

the enzyme. For each 10° increase in temperature, the Enzymatic reactions may not progress normally if a par-
rate of the reaction will approximately double until, of ticular substance, an inhibitor, interferes with the reac-
course, the protein is denatured. tion. Competitive inhibitors physically bind to the active
Each enzyme functions optimally at a particular tem- site of an enzyme and compete with the substrate for the
perature, which is influenced by other reaction variables, active site. With a substrate concentration significantly
especially the total time for the reaction. The optimal higher than the concentration of the inhibitor, the inhi-
temperature is usually close to that of the physiologic bition is reversible because the substrate is more likely
environment of the enzyme; however, some denaturation than the inhibitor to bind the active site and the enzyme
may occur at the human physiologic temperature of has not been destroyed.
37°C. The rate of denaturation increases as the tempera- A noncompetitive inhibitor binds an enzyme at a place
ture increases and is usually significant at 40°C to 50°C. other than the active site and may be reversible in that
Because low temperatures render enzymes reversibly some naturally present metabolic substances combine
inactive, many serum or plasma specimens for enzyme reversibly with certain enzymes. Noncompetitive inhi-
measurement are refrigerated or frozen to prevent activ- bition also may be irreversible if the inhibitor destroys
ity loss until analysis. Storage procedures may vary from part of the enzyme involved in catalytic activity. Because
enzyme to enzyme because of individual stability charac- the inhibitor binds the enzyme independently from the
teristics. Repeated freezing and thawing, however, tends substrate, increasing substrate concentration does not
to denature protein and should be avoided. reverse the inhibition.
Because of their temperature sensitivity, enzymes Uncompetitive inhibition is another kind of inhibition in
should be analyzed under strictly controlled temperature which the inhibitor binds to the ES complex—increasing
conditions. Incubation temperatures should be accurate substrate concentration results in more ES complexes
within ±0.1°C. Laboratories usually attempt to establish to which the inhibitor binds and, thereby, increases the
an analysis temperature for routine enzyme measure- inhibition. The enzyme–substrate–inhibitor complex
ment of 25°C, 30°C, or 37°C. Attempts to establish a does not yield product.
universal temperature for enzyme analysis have been Each of the three kinds of inhibition is unique with
unsuccessful, and therefore, reference ranges for enzyme respect to effects on the Vmax and Km of enzymatic reac-
levels may vary significantly among laboratories. In the tions (Fig. 13-4). In competitive inhibition, the effect
United States, however, 37°C is most commonly used. of the inhibitor can be counteracted by adding excess
Cofactors substrate to bind the enzyme. The amount of the inhibi-
Cofactors are nonprotein entities that must bind to tor is then negligible by comparison, and the reaction
particular enzymes before a reaction occurs. Common will proceed at a slower rate but to the same maximum
activators (inorganic cofactors) are metallic (Ca2+, Fe2+, velocity as an uninhibited reaction. Km is a constant for
Mg2+, Mn2+, Zn2+, and K+) and nonmetallic (Br− and each enzyme and cannot be altered. However, because
Cl–). The activator may be essential for the reaction or the amount of substrate needed to achieve a particular
may only enhance the reaction rate in proportion with velocity is higher in the presence of a competing inhibi-
concentration to the point at which the excess activa- tor, Km appears to increase when exhibiting the effect of
tor begins to inhibit the reaction. Activators function by the inhibitor.
alternating the spatial configuration of the enzyme for The substrate and inhibitor, commonly a metallic
proper substrate binding, linking substrate to the enzyme ion, may bind an enzyme simultaneously in noncom-
or coenzyme, or undergoing oxidation or reduction. petitive inhibition. The inhibitor may inactivate either
Some common coenzymes (organic cofactors) are an ES complex or just the enzyme by causing struc-
nucleotide phosphates and vitamins. Coenzymes serve as tural changes in the enzyme. Even if the inhibitor binds
second substrates for enzymatic reactions. When bound reversibly and does not inactivate the enzyme, the pres-
tightly to the enzyme, coenzymes are called prosthetic ence of the inhibitor when it is bound to the enzyme
groups. For example, NAD as a cofactor may be reduced slows the rate of the reaction. Thus, for noncompetitive
to nicotinamide adenine dinucleotide phosphate (NADP) inhibition, the maximum reaction velocity cannot be
in a reaction in which the primary substrate is oxidized. achieved. Increasing substrate levels have no influence
Increasing coenzyme concentration will increase the on the binding of a noncompetitive inhibitor, so that Km
velocity of an enzymatic reaction in a manner synony- is unchanged.
mous with increasing substrate concentration. When Because uncompetitive inhibition requires the forma-
quantitating an enzyme that requires a particular cofac- tion of an ES complex, increasing substrate concentra-
tor, that cofactor should always be provided in excess so tion increases inhibition. Therefore, maximum velocity
that the extent of the reaction does not depend on the equal to that of an uninhibited reaction cannot be
concentration of the cofactor. achieved, and Km appears to be decreased.
268 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

Vmax Vmax
Vmax

Km Km Km
[S] [S] [S]

FIGURE 13-4 Normal Lineweaver-Burk plot (solid line) compared with each type of enzyme inhibition (dotted line). (A) Competitive inhibi-
tion Vmax unaltered; Km appears increased. (B) Noncompetitive inhibition Vmax decreased; Km unchanged. (C) Uncompetitive inhibition
Vmax decreased; Km appears decreased.

Measurement of Enzyme Activity the substrate on which an intermediate auxiliary enzyme

acts. A product of the intermediate reaction becomes the
Because enzymes are usually present in very small quan-
substrate for the final reaction, which is catalyzed by an
tities in biologic fluids and often difficult to isolate from
indicator enzyme and commonly involves the conversion
similar compounds, a convenient method of enzyme
of NAD to NADH or vice versa.
quantitation is measurement of catalytic activity. Activity When performing an enzyme quantitation in zero-
is then related to concentration. Common methods order kinetics, inhibitors must be lacking and other
might photometrically measure an increase in product variables that may influence the rate of the reaction must
concentration, a decrease in substrate concentration, a be carefully controlled. A constant pH should be main-
decrease in coenzyme concentration, or an increase in tained by means of an appropriate buffer solution. The
the concentration of an altered coenzyme. temperature should be constant within ±0.1°C through-
If the amount of substrate and any coenzyme is in out the assay at a temperature at which the enzyme is
excess in an enzymatic reaction, the amount of substrate active (usually, 25°C, 30°C, or 37°C).
or coenzyme used, or product or altered coenzyme During the progress of the reaction, the period for
formed, will depend only on the amount of enzyme the analysis also must be carefully selected. When the
present to catalyze the reaction. Enzyme concentrations, enzyme is initially introduced to the reactants and the
therefore, are always performed in zero-order kinetics, excess substrate is steadily combining with the avail-
with the substrate in sufficient excess to ensure that no able enzyme, the reaction rate rises. After the enzyme is
more than 20% of the available substrate is converted to saturated, the rates of product formation, release of the
product. Any coenzyme also must be in excess. NADH enzyme, and recombination with more substrate proceed
is a coenzyme frequently measured in the laboratory. linearly. After some time, usually 6 to 8 minutes after
NADH absorbs light at 340 nm, whereas NAD does reaction initiation, the reaction rate decreases as the
not, and a change in absorbance at 340 nm is easily substrate is depleted, the reverse reaction occurs appre-
measured. ciably, and the product begins to inhibit the reaction.
In specific laboratory methodologies, substances Hence, enzyme quantitations must be performed during
other than substrate or coenzyme are necessary and the linear phase of the reaction.
must be present in excess. NAD or NADH is often con- One of two general methods may be used to measure
venient as a reagent for a coupled-enzyme assay when the extent of an enzymatic reaction: (1) fixed-time and
neither NAD nor NADH is a coenzyme for the reaction. (2) continuous-monitoring or kinetic assay. In the fixed-
In other coupled-enzyme assays, more than one enzyme time method, the reactants are combined, the reaction
is added in excess as a reagent and multiple reactions proceeds for a designated time, the reaction is stopped
are catalyzed. After the enzyme under analysis catalyzes (usually by inactivating the enzyme with a weak acid),
its specific reaction, a product of that reaction becomes and a measurement of the amount of reaction that has
 CHAPTER 13 n ENZYMES 269

occurred is made. The reaction is assumed to be linear of Units (Système International d’Unités [SI]) is the
over the reaction time; the larger the reaction, the more katal (mol/s). The mole is the unit for substrate concen-
the enzyme present. tration, and the unit of time is the second. Enzyme con-
In continuous-monitoring or kinetic assays, multiple centration is then expressed as katals per liter (kat/L)
measurements, usually of absorbance change, are made (1.0 IU = 17 nkat).
during the reaction, either at specific time intervals When enzymes are quantitated by measuring the
(usually every 30 or 60 seconds) or continuously by a increase or decrease of NADH at 340 nm, the molar
continuous-recording spectrophotometer. These assays absorptivity (6.22 × 103 mol/L) of NADH is used to cal-
are advantageous over fixed-time methods because culate enzyme activity.
the linearity of the reaction may be more adequately
verified. If absorbance is measured at intervals, several Measurement of Enzyme Mass
data points are necessary to increase the accuracy of
linearity assessment. Continuous measurements are Immunoassay methodologies that quantify enzyme
preferred because any deviation from linearity is readily concentration by mass are also available and are
observable. routinely used for quantification of some enzymes,
The most common cause of deviation from linearity such as creatine kinase (CK)-MB. Immunoassays may
occurs when the enzyme is so elevated that all substrate overestimate active enzyme as a result of possible
is used early in the reaction time. For the remainder cross-reactivity with inactive enzymes, such as zymo-
of the reaction, the rate change is minimal, with the gens, inactive isoenzymes, macroenzymes, or partially
implication that the coenzyme concentration is very digested enzyme. The relationship between enzyme
low. With continuous monitoring, the laboratorian may activity and enzyme quantity is generally linear but
observe a sudden decrease in the reaction rate (devia- should be determined for each enzyme. Enzymes may
tion from zero-order kinetics) of a particular determi- also be determined and quantified by electrophoretic
nation and may repeat the determination using less techniques, which provide resolution of isoenzymes
patient sample. The decrease in the amount of patient and isoforms.
sample operates as a dilution, and the answer obtained Ensuring the accuracy of enzyme measurements
may be multiplied by the dilution factor to obtain the has long been a concern of laboratorians. The Clinical
final answer. The sample itself is not diluted so that Laboratory Improvement Amendment of 1988 (CLIA
the diluent cannot interfere with the reaction. (Sample ’88) has established guidelines for quality control and
dilution with saline may be necessary to minimize nega- proficiency testing for all laboratories. Problems with
tive effects in analysis caused by hemolysis or lipemia.) quality control materials for enzyme testing have been
Enzyme activity measurements may not be accurate if a significant issue. Differences between clinical speci-
storage conditions compromise integrity of the protein, mens and control sera include species of origin of the
if enzyme inhibitors are present, or if necessary cofac- enzyme, integrity of the molecular species, isoenzyme
tors are not present. forms, matrix of the solution, addition of preservatives,
and lyophilization processes. Many studies have been
Calculation of Enzyme Activity conducted to ensure accurate enzyme measurements and
good quality control materials.4
When enzymes are quantified relative to their activity
rather than a direct measurement of concentration, the
Enzymes as Reagents
units used to report enzyme levels are activity units.
The definition for the activity unit must consider vari- Enzymes may be used as reagents to measure many
ables that may alter results (e.g., pH, temperature, and nonenzymatic constituents in serum. For example, glu-
substrate). Historically, specific method developers cose, cholesterol, and uric acid are frequently quantified
frequently established their own units for reporting by means of enzymatic reactions, which measure the
results and often named the units after themselves (i.e., concentration of the analyte due to the specificity of the
Bodansky and King units). To standardize the system enzyme. Enzymes are also used as reagents for methods
of reporting quantitative results, the EC defined the to quantify analytes that are substrates for the corre-
international unit (IU) as the amount of enzyme that sponding enzyme. One example, lactate dehydrogenase
will catalyze the reaction of 1 µmol of substrate per (LD), may be a reagent when lactate or pyruvate concen-
minute under specified conditions of temperature, pH, trations are evaluated. For such methods, the enzyme
substrates, and activators. Since specified conditions is added in excess in a quantity sufficient to provide a
may vary among laboratories, reference values are still complete reaction in a short period.
often laboratory specific. Enzyme concentration is Immobilized enzymes are chemically bonded to adsor-
usually expressed in units per liter (IU/L). The unit of bents, such as agarose or certain types of cellulose, by
enzyme activity recognized by the International System azide groups, diazo, and triazine. The enzymes act as
270 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

recoverable reagents. When substrate is passed through peroxidase, alkaline phosphatase (ALP), glucose-6-phos-
the preparation, the product is retrieved and analyzed, phate dehydrogenase (G-6-PD), and β-galactosidase.
and the enzyme is present and free to react with more The enzyme in these assays functions as an indicator that
substrate. Immobilized enzymes are convenient for reflects either the presence or the absence of the analyte.
batch analyses and are more stable than enzymes in a
solution. Enzymes are also commonly used as reagents
ENZYMES OF CLINICAL SIGNIFICANCE
in competitive and noncompetitive immunoassays, such
as those used to measure human immunodeficiency Table 13-2 lists the commonly analyzed enzymes, includ-
virus antibodies, therapeutic drugs, and cancer anti- ing their systematic names and clinical significance. Each
gens. Commonly used enzymes include horseradish enzyme is discussed in this chapter with respect to tissue

TABLE 13-2 MAJOR ENZYMES OF CLINICAL SIGNIFICANCE

ENZYME CLINICAL SIGNIFICANCE
Acid phosphatase (ACP) Prostatic carcinoma
Alanine aminotransferase (ALT) Hepatic disorder
Aldolase (ALD) Skeletal muscle disorder
Alkaline phosphatase (ALP) Hepatic disorder
Bone disorder
Amylase (AMY) Acute pancreatitis
Angiotensin-converting enzyme (ACE) Blood pressure regulation
Aspartate aminotransferase (AST) Myocardial infarction
Hepatic disorder
Skeletal muscle disorder
Chymotrypsin (CHY) Chronic pancreatitis insufficiency
Creatine kinase (CK) Myocardial infarction
Skeletal muscle disorder
Elastase-1 (E1) Chronic pancreatitis insufficiency
Glucose-6-phosphate dehydrogenase (G-6-PD) Drug-induced hemolytic anemia
Glutamate dehydrogenase (GLD) Hepatic disorder
γ-Glutamyltransferase (GGT) Hepatic disorder
Glutathione-S-transferase (GST) Hepatic disorder
Glycogen phosphorylase (GP) Acute myocardial infarction
Lactate dehydrogenase (LD) Myocardial infarction
Hepatic disorder
Hemolysis
Carcinoma
Lipase (LPS) Acute pancreatitis
5′-Nucleotidase Hepatic disorder
Pseudocholinesterase (PChE) Organophosphate poisoning
Genetic variants
Hepatic disorder
Suxamethonium sensitivity
Pyruvate kinase (PK) Hemolytic anemia
Trypsin (TRY) Acute pancreatitis
 CHAPTER 13 n ENZYMES 271

Diagnostic Significance
CASE STUDY 13-1 Due to the high concentrations of CK in muscle tis-
A 51-year-old, overweight white man visits his family sue, CK levels are frequently elevated in disorders of
physician with a symptom of “indigestion” of 5 days’ cardiac and skeletal muscle (myocardial infarction [MI],
duration. He has also had bouts of sweating, malaise, rhabdomyolysis, and muscular dystrophy). The CK level
and headache. His blood pressure is 140/105 mm is considered a sensitive indicator of acute myocardial
Hg; his family history includes a father with diabetes infarction (AMI) and muscular dystrophy, particularly
who died at age 62 of AMI secondary to diabetes the Duchenne type. Extreme elevations of CK occur in
mellitus. An electrocardiogram revealed changes Duchenne-type muscular dystrophy, with values reach-
from one performed 6 months earlier. The results of ing 50 to 100 times the upper limit of normal (ULN).
the patient’s blood work are as follows: Although total CK levels are sensitive indicators of these
disorders, they are not entirely specific indicators as CK
CK 129 U/L (30–60) elevation is found in various other abnormal cardiac and
skeletal muscle conditions. Levels of CK also vary with
CK-MB 4% (<6%)
muscle mass and, therefore, may depend on gender, race,
LD 280 U/L (100–225) degree of physical conditioning, and age.
LD Isoenzymes LD-1 > LD-2 Elevated CK levels are also occasionally seen in cen-
AST 35 U/L (5–30) tral nervous system disorders such as strokes, seizures,
nerve degeneration, and central nervous system shock.
Questions Damage to the blood–brain barrier must occur to allow
enzyme release to the peripheral circulation.
1. Can a diagnosis of AMI be ruled out in this
Other pathophysiologic conditions in which elevated
patient?
CK levels occur are hypothyroidism, malignant hyper-
2. What further cardiac markers should be run on pyrexia, and Reye’s syndrome. Table 13-3 lists the major
this patient? disorders associated with abnormal CK levels. Serum CK
levels and CK/progesterone ratio have been useful in the
3. Should this patient be admitted to the hospital?
diagnosis of ectopic pregnancies.5 Total serum CK levels
have also been used as an early diagnostic tool to identify
patients with Vibrio vulnificus infections.6
Because enzyme elevation is found in numerous dis-
source, diagnostic significance, assay method, source of orders, the separation of total CK into its various isoen-
error, and reference range. zyme fractions is considered a more specific indicator of
various disorders than total levels. Typically, the clinical
Creatine Kinase relevance of CK activity depends more on isoenzyme
CK is an enzyme with a molecular weight of approxi- fractionation than on total levels.
mately 82,000 Da that is generally associated with ATP CK occurs as a dimer consisting of two subunits that
regeneration in contractile or transport systems. Its can be separated readily into three distinct molecular forms.
predominant physiologic function occurs in muscle The three isoenzymes have been designated as CK-BB (brain
cells, where it is involved in the storage of high-energy type), CK-MB (hybrid type), and CK-MM (muscle type).
creatine phosphate. Every contraction cycle of muscle On electrophoretic separation, CK-BB will migrate fastest
results in creatine phosphate use, with the production of toward the anode and is therefore called CK-1. CK-BB is
ATP. This results in relatively constant levels of muscle followed by CK-MB (CK-2) and, finally, by CK-MM (CK-
ATP. The reversible reaction catalyzed by CK is shown 3), exhibiting the slowest mobility (Fig. 13-5). Table 13-3
in Equation 13-4: indicates the tissue localization of the isoenzymes and the
CK
major conditions associated with elevated levels. Separation
Creatine + ATP ∆ Creatine phosphate + ADP of CK isoforms may also be visualized by high-voltage elec-
(Eq. 13-4) trophoretic separation. Isoforms occur following cleavage
of the carboxyl-terminal amino acid from the M subunit
Tissue Source by serum carboxypeptidase N. Three isoforms have been
CK is widely distributed in tissue, with highest activities described for CK-MM and two isoforms for CK-MB; the
found in skeletal muscle, heart muscle, and brain tissue. clinical significance is not well established.
CK is present in much smaller quantities in other tissue The major isoenzyme in the sera of healthy people is
sources, including the bladder, placenta, gastrointestinal the MM form. Values for the MB isoenzyme range from
tract, thyroid, uterus, kidney, lung, prostate, spleen, undetectable to trace (<6% of total CK). It also appears
liver, and pancreas. that CK-BB is present in small quantities in the sera of
272 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

CREATINE KINASE ISOENZYMES—TISSUE LOCALIZATION AND SOURCES

TABLE 13-3
OF ELEVATION
ISOENZYME TISSUE CONDITION
CK-MM Heart Myocardial infarction
Skeletal muscle Skeletal muscle disorder
Muscular dystrophy
Polymyositis
Hypothyroidism
Malignant hyperthermia
Physical activity
Intramuscular injection
CK-MB Heart Myocardial infarction
Skeletal muscle Myocardial injury
Ischemia
Angina
Inflammatory heart disease
Cardiac surgery
Duchenne-type muscular dystrophy
Polymyositis
Malignant hyperthermia
Reye’s syndrome
Rocky Mountain spotted fever
Carbon monoxide poisoning
CK-BB Brain Central nervous system shock
Bladder Anoxic encephalopathy
Lung Cerebrovascular accident
Prostate Seizure
Uterus Placental or uterine trauma
Colon Carcinoma
Stomach Reye’s syndrome
Thyroid Carbon monoxide poisoning
Malignant hyperthermia
Acute and chronic renal failure

healthy people; however, the presence of CK-BB in serum (Table 13-3). Hypothyroidism results in CK-MM ele-
depends on the method of detection. Most techniques vations because of the involvement of muscle tissue
cannot detect CK-BB in normal serum. (increased membrane permeability), the effect of thyroid
CK-MM is the major isoenzyme fraction found in stri- hormone on enzyme activity, and, possibly, the slower
ated muscle and normal serum. Skeletal muscle contains clearance of CK as a result of slower metabolism.
almost entirely CK-MM, with a small amount of CK-MB. Mild to strenuous activity may contribute to elevated
The majority of CK activity in heart muscle is also attrib- CK levels, as may intramuscular injections. In physical
uted to CK-MM, with approximately 20% as a result of activity, the extent of elevation is variable. However,
CK-MB.7 Normal serum consists of approximately 94% to the degree of exercise in relation to the exercise capac-
100% CK-MM. Injury to both cardiac and skeletal muscle ity of the individual is the most important factor in
accounts for the majority of cases of CK-MM elevations determining the degree of elevation.8 Patients who are
 CHAPTER 13 n ENZYMES 273

findings indicate that CK-BB may be a useful tumor-

associated marker.9 The most common causes of CK-BB
elevations are central nervous system damage, tumors,
childbirth, and the presence of macro-CK, an enzyme–
immunoglobulin complex. In most of these cases, the
CK-BB level is greater than 5 U/L, usually in the range of
FIGURE 13-5  Electrophoretic migration pattern of normal and
atypical creatine kinase (CK) isoenzymes.
10 to 50 U/L. Other conditions listed in Table 13-3 usu-
ally show CK-BB activity below 10 U/L.10
The value of CK isoenzyme separation can be found
physically well conditioned show lesser degrees of eleva- principally in the detection of myocardial damage.
tion than do patients who are less conditioned. Levels Cardiac tissue contains significant quantities of CK-MB,
may be elevated for as long as 48 hours following exer- approximately 20% of all CK-MB. Whereas CK-MB is
cise. CK elevations are generally less than five times the found in small quantities in other tissue, myocardium is
ULN following intramuscular injections and usually not essentially the only tissue from which CK-MB enters the
apparent after 48 hours, although elevations may persist serum in significant quantities. Demonstration of elevat-
for 1 week. The predominant isoenzyme is CK-MM. ed levels of CK-MB, greater than or equal to 6% of the
The quantity of CK-BB in the tissue (Table 13-3) total CK, is considered a good indicator of myocardial
is usually small. The small quantity, coupled with its damage, particularly AMI. Other nonenzyme proteins,
relatively short half-life (1 to 5 hours), results in CK-BB called troponins, have been found to be even more spe-
activities that are generally low and transient and not cific and may elevate in the absence of CK-MB elevations.
usually measurable when tissue damage occurs. Highest Following MI, the CK-MB levels begin to rise within 4
concentrations are found in the central nervous system, to 8 hours, peak at 12 to 24 hours, and return to normal
the gastrointestinal tract, and the uterus during preg- levels within 48 to 72 hours. This time frame must be
nancy. Although brain tissue has high concentrations considered when interpreting CK-MB levels.
of CK, serum rarely contains CK-BB of brain origin. CK-MB activity has been observed in other cardiac
Because of its molecular size (80,000 Da), its passage disorders (Table 13-3). Therefore, increased quantities
across the blood–brain barrier is hindered. However, are not entirely specific for AMI but probably reflect
when extensive damage to the brain has occurred, sig- some degree of ischemic heart damage. The specificity of
nificant amounts of CK-BB can sometimes be detected in CK-MB levels in the diagnosis of AMI can be increased
the serum. It has been observed that CK-BB may be sig- if interpreted in conjunction with LD isoenzymes and/or
nificantly elevated in patients with carcinoma of various troponins and if measured sequentially over a 48-hour
organs. It has been found in association with untreated period to detect the typical rise and fall of enzyme activ-
prostatic carcinoma and other adenocarcinomas. These ity seen in AMI (Fig. 13-6). The MB isoenzyme also has

10 CK-MB
Degree of elevation (× ULN)

6 CK

3 AST
LD

1 2 3 4 5 6 10
Time course of elevation (days)
FIGURE 13-6  Time-activity curves of enzymes in myocardial infarction for aspartate aminotransferase
(AST), creatine kinase (CK), CK-MB, and lactate dehydrogenase (LD). CK, specifically the MB fraction,
increases initially, followed by AST and LD. LD is elevated the longest. All enzymes usually return to normal
within 10 d. ULN, upper limit of normal.
274 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

been detected in the sera of patients with noncardiac and several immunoassays, including radioimmunoassay
disorders. CK-MB levels found in these conditions prob- (RIA) and immunoinhibition methods. Although mass
ably represent leakage from skeletal muscle, although in methods are more sensitive and preferred for quantita-
Duchenne-type muscular dystrophy, there may be some tion of CK-MB, electrophoresis has been the reference
cardiac involvement as well. CK-MB levels in Reye’s syn- method. The electrophoretic properties of the CK isoen-
drome also may reflect myocardial damage. Despite the zymes are shown in Figure 13-5. Generally, the technique
findings of CK-MB levels in disorders other than MI, its consists of performing electrophoresis on the sample,
presence still remains a significant indicator of AMI.11 measuring the reaction using an overlay technique,
The typical time course of CK-MB elevation following and then visualizing the bands under ultraviolet light.
AMI is not found in other conditions. With electrophoresis, the atypical bands can be sepa-
Nonenzyme proteins (troponin I and troponin T) rated, allowing their detection apart from the three major
have been used as a more sensitive and specific marker of bands. Often a strongly fluorescent band appears, which
myocardial damage. These proteins are released into the migrates in close proximity to the CK-BB form. The exact
bloodstream earlier and persist longer than CK and its nature of this fluorescence is unknown, but it has been
isoenzyme CK-MB. More information on these protein attributed to the binding of fluorescent drugs or bilirubin
markers of AMI can be found in Chapter 26. by albumin.
Numerous reports have been made describing the In addition to visualizing atypical CK bands, other
appearance of unusual CK isoenzyme bands display- advantages of electrophoresis methods include detecting
ing electrophoretic properties that differ from the three an unsatisfactory separation and allowing visualization
major isoenzyme fractions (Fig. 13-5).12-16 These atypi- of adenylate kinase (AK). AK is an enzyme released from
cal forms are generally of two types and are referred to as erythrocytes in hemolyzed samples and appearing as a
macro-CK and mitochondrial CK (CK-Mi). band cathodal to CK-MM. AK may interfere with chemi-
Macro-CK appears to migrate to a position midway cal or immunoinhibition methods, causing a falsely
between CK-MM and CK-MB. This type of macro-CK elevated CK or CK-MB value.
largely comprises CK-BB complexed with immunoglobu- Ion-exchange chromatography has the potential for
lin. In most instances, the associated immunoglobulin being more sensitive and precise than electrophoretic
is IgG, although a complex with IgA also has been procedures performed with good technique. On an
described. The term macro-CK has also been used to unsatisfactory column, however, CK-MM may merge
describe complexes of lipoproteins with CK-MM. The into CK-MB and CK-BB may be eluted with CK-MB.
incidence of macro-CK in sera ranges from 0.8% to 1.6%. Also, macro-CK may elute with CK-MB.
Currently, no specific disorder is associated with its Antibodies against both the M and B subunits have
presence, although it appears to be age and sex related, been used to determine CK-MB activity. Anti-M inhibits
occurring most frequently in women older than age 50. all M activity but not B activity. CK activity is measured
CK-Mi is bound to the exterior surface of the inner before and after inhibition. Activity remaining after M
mitochondrial membranes of muscle, brain, and liver. inhibition is a result of the B subunit of both MB and
It migrates to a point cathodal to CK-MM and exists as BB activity. The residual activity after inhibition is mul-
a dimeric molecule of two identical subunits. It occurs tiplied by 2 to account for MB activity (50% inhibited).
in serum in both the dimeric state and in the form of The major disadvantage of this method is that it detects
oligomeric aggregates of high molecular weight (350,000 BB activity, which, although not normally detectable,
Da). CK-Mi is not present in normal serum and is typi- will cause falsely elevated MB results when BB is present.
cally not present following MI. The incidence of CK-Mi In addition, the atypical forms of CK-Mi and macro-CK
ranges from 0.8% to 1.7%. For it to be detected in serum, are not inhibited by anti-M antibodies and also may
extensive tissue damage must occur, causing breakdown cause erroneous results for MB activity.
of the mitochondrion and cell wall. Its presence does not Immunoassays detect CK-MB reliably with minimal
correlate with any specific disease state but appears to be cross-reactivity. Immunoassays measure the concentra-
an indicator of severe illness. CK-Mi has been detected tion of enzyme protein rather than enzymatic activity
in cases of malignant tumor and cardiac abnormalities. and can, therefore, detect enzymatically inactive CK-MB.
In view of the indefinite correlation between these This leads to the possibility of permitting detection of
atypical CK forms and a specific disease state, it appears infarction earlier than other methods. A double-antibody
that their significance relates primarily to the methods immunoinhibition assay is also available. This technique
used for detecting CK-MB. In certain analytic proce- allows differentiation of MB activity due to AK and the
dures, these atypical forms may be measured as CK-MB, atypical isoenzymes, resulting in a more specific analytic
resulting in erroneously high CK-MB levels. procedure for CK-MB.17 Point-of-care assay systems for
Methods used for the measurement of CK isoenzymes CK-MB are available but not as widely used as those for
include electrophoresis, ion-exchange chromatography, troponins.
 CHAPTER 13 n ENZYMES 275

Assay Enzyme Activity Reference Range

As indicated by Equation 13-4, CK catalyzes both for- Total CK:
ward and reverse reactions involving phosphorylation of
Males: 46 to 171 U/L (37°C) (0.8 to 2.9 µkat/L)
creatine or ADP. Typically, for analysis of CK activity,
Females: 34 to 145 U/L (37°C) (0.6 to 2.4 µkat/L)
this reaction is coupled with other enzyme systems and a
CK-MB: <5% total CK
change in absorbance at 340 nm is determined. The for-
ward reaction is coupled with the pyruvate kinase–LD– The higher values in males are attributed to increased
NADH system and proceeds according to Equation 13-5: muscle mass. Note that enzyme reference ranges are
CK subject to variation, depending on the method used and
Creatine + ATP ∆ Creatine phosphate + ADP
the assay conditions.
PK
ADP + phosphoenolpyruvate ∆ Pyruvate + ATP
Lactate Dehydrogenase
Pyruvate + NADH + H+ ∆ Lactate + NAD+
LD

(Eq. 13-5) LD is an enzyme that catalyzes the interconversion of

lactic and pyruvic acids. It is a hydrogen-transfer enzyme
The reverse reaction is coupled with the hexokinase– that uses the coenzyme NAD+ according to Equation 13-7:
G-6-PD–NADP system, as indicated in Equation 13-6:
CK CH3 CH3
Creatine phosphate + ADP ∆ Creatine ADP
KH LD
ATP + glucose ∆ ADP + glucose-6-phosphate HCM OH NAD CBO NADH H
G-6-PD
Glucose 6-phosphate + NADPH+ ∆
COOH COOH
6-phosphogluconate + NADPH (Eq. 13-6)
Lactate Pyruvate
The reverse reaction proposed by Oliver and modified (Eq. 13-7)
by Rosalki is the most commonly performed method in
the clinical laboratory.13 The reaction proceeds two to Tissue Source
six times faster than the forward reaction, depending on LD is widely distributed in the body. High activities
the assay conditions and there is less interference from are found in the heart, liver, skeletal muscle, kidney,
side reactions. The optimal pH for the reverse reaction is and erythrocytes; lesser amounts are found in the lung,
6.8; for the forward reaction, it is 9.0. smooth muscle, and brain.
CK activity in serum is unstable, being rapidly
inactivated because of oxidation of sulfhydryl groups. Diagnostic Significance
Inactivation can be partially reversed by the addition of Because of its widespread activity in numerous body tis-
sulfhydryl compounds to the assay reagent. Compounds sue, LD is elevated in a variety of disorders. Increased
such as N-acetylcysteine, mercaptoethanol, thioglycerol, levels are found in cardiac, hepatic, and skeletal muscle
and dithiothreitol are among those used. and renal diseases, as well as in several hematologic
and neoplastic disorders. The highest levels of total
Source of Error LD are seen in pernicious anemia and hemolytic disor-
Hemolysis of serum samples may be a source of elevated ders. Intramedullary destruction of erythroblasts causes
CK activity. Erythrocytes are virtually devoid of CK; elevation as a result of the high concentration of LD in
however, they are rich in AK activity. AK reacts with erythrocytes. Liver disorders, such as viral hepatitis and
ADP to produce ATP, which is then available to par- cirrhosis, show slight elevations of two to three times the
ticipate in the assay reaction, causing falsely elevated ULN. AMI and pulmonary infarct also show slight eleva-
CK levels. This interference can occur with hemolysis of tions of approximately the same degree (2 to 3× ULN).
greater than 320 mg/L hemoglobin, which releases suf- In AMI, LD levels begin to rise within 12 to 24 hours,
ficient AK to exhaust the AK inhibitors in the reagent. reach peak levels within 48 to 72 hours, and may remain
Trace hemolysis causes little, if any, CK elevation. Serum elevated for 10 days. Skeletal muscle disorders and some
should be stored in a dark place because CK is inactivat- leukemias contribute to increased levels. Marked eleva-
ed by light. Activity can be restored after storage in the tions can be observed in most patients with acute lym-
dark at 4°C for 7 days or at −20°C for 1 month when the phoblastic leukemia in particular.
assay is conducted using a sulfhydryl activator.18 Because Because of the many conditions that contribute to
of the effect of muscular activity and muscle mass on CK increased activity, an elevated total LD value is a rather
levels, it should be noted that people who are physically nonspecific finding. LD assays, therefore, assume more
well trained tend to have elevated baseline levels and that clinical significance when separated into isoenzyme frac-
patients who are bedridden for prolonged periods may tions. The enzyme can be separated into five major frac-
have decreased CK activity. tions, each comprising four subunits. It has a molecular
276 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

LACTATE DEHYDROGENASE ISOENZYMES—TISSUE LOCALIZATION AND SOURCES

TABLE 13-4
OF ELEVATION
ISOENZYME TISSUE DISORDER
LD-1 (HHHH) Heart Myocardial infarction
Red blood cells Hemolytic anemia
LD-2 (HHHM) Heart Megaloblastic anemia
Red blood cells Acute renal infarct
Hemolyzed specimen
LD-3 (HHMM) Lung Pulmonary embolism
Lymphocytes Extensive
Spleen Pulmonary pneumonia
Pancreas Lymphocytosis
Acute pancreatitis
Carcinoma
LD-4 (HMMM) Liver Hepatic injury or inflammation
LD-5 (MMMM) Skeletal muscle Skeletal muscle injury
LD, lactate dehydrogenase.

weight of 128,000 Da. Each isoenzyme comprises four clinical significance in the detection of hepatic disorders,
polypeptide chains with a molecular weight of 32,000 particularly intrahepatic disorders. Disorders of skeletal
Da each. Two different polypeptide chains, designated H muscle will reveal elevated LD-5 levels, as depicted in the
(heart) and M (muscle), combine in five arrangements muscular dystrophies.
to yield the five major isoenzyme fractions. Table 13-4 A sixth LD isoenzyme has been identified, which
indicates the tissue localization of the LD isoenzymes migrates cathodic to LD-5.21-23 LD-6 is alcohol dehydro-
and the major disorders associated with elevated levels. genase. In reporting studies, LD-6 has been present in
LD-1 migrates most quickly toward the anode, followed patients with arteriosclerotic cardiovascular failure. It is
in sequence by the other fractions, with LD-5 migrating believed that its appearance signifies a grave prognosis
the slowest. and impending death. LD-5 is elevated concurrently with
In the sera of healthy individuals, the major isoen- the appearance of LD-6, probably representing hepatic
zyme fraction is LD-2, followed by LD-1, LD-3, LD-4, congestion due to cardiovascular disease. It is suggested,
and LD-5 (for the isoenzyme ranges, see Table 13-5). therefore, that LD-6 may reflect liver injury secondary to
LD-1 and LD-2 are present to approximately the same severe circulatory insufficiency.
extent in the tissues listed in Table 13-4. However,
cardiac tissue and red blood cells contain a higher con-
centration of LD-1. Therefore, in conditions involving
cardiac necrosis (AMI) and intravascular hemolysis, the LACTATE DEHYDROGENASE
serum levels of LD-1 will increase to a point at which TABLE 13-5 (LD) ISOENZYMES AS A
PERCENTAGE OF TOTAL LD
they are present in greater concentration than LD-2,
resulting in a condition known as the LD flipped pat- ISOENZYME %
tern (LD-1 > LD-2).19 This flipped pattern is suggestive LD-1 14–26
of AMI. However, LD is not specific to cardiac tissue
and is not a preferred marker of diagnosis of AMI. LD-1/ LD-2 29–39
LD-2 ratios greater than 1 also may be observed in LD-3 20–26
hemolyzed serum samples.20 Elevations of LD-3 occur LD-4 8–16
most frequently with pulmonary involvement and are LD-5 6–16
also observed in patients with various carcinomas. The
LD-4 and LD-5 isoenzymes are found primarily in liver Source: Lott JA, Stang JM. Serum enzymes and isoenzymes in the
diagnosis and differential diagnosis of myocardial ischemia and
and skeletal muscle tissue, with LD-5 being the predomi- necrosis. Clin Chem. 1980;26:1241.
nant fraction in these tissues. LD-5 levels have greatest
 CHAPTER 13 n ENZYMES 277

LD has been shown to complex with immunoglob- However, the reverse reaction is more susceptible to
ulins and to reveal atypical bands on electrophoresis. substrate exhaustion and loss of linearity. The optimal
LD complexed with IgA and IgG usually migrates pH for the forward reaction is 8.3 to 8.9; for the reverse
between LD-3 and LD-4. This macromolecular com- reaction, it is 7.1 to 7.4.
plex is not associated with any specific clinical abnor-
mality. Source of Error
Analysis of LD isoenzymes can be accomplished Erythrocytes contain an LD concentration approximately
by electrophoresis, by immunoinhibition or chemi- 100 to 150 times that found in serum. Therefore, any
cal inhibition methods, or by differences in substrate degree of hemolysis should render a sample unaccept-
affinity. Because of limited clinical utility, such tests able for analysis. LD activity is unstable in serum regard-
are not commonly used. The electrophoretic proce- less of the temperature at which it is stored. If the sample
dure has been widely used historically. After electro- cannot be analyzed immediately, it should be stored at
phoretic separation, the isoenzymes can be detected 25°C and analyzed within 48 hours. LD-5 is the most
either fluorometrically or colorimetrically. LD can labile isoenzyme. Loss of activity occurs more quickly
use other substrates in addition to lactate, such as at 4°C than at 25°C. Serum samples for LD isoenzyme
α-hydroxybutyrate. The H subunits have a greater affin- analysis should be stored at 25°C and analyzed within
ity for α-hydroxybutyrate than to the M subunits. This 24 hours of collection.
has led to the use of this substrate in an attempt to mea-
sure the LD-1 activity, which consists entirely of H sub- Reference Range
units. The chemical assay, known as the measurement LD, 125 to 220 U/L (37°C)
of α-hydroxybutyrate dehydrogenase activity (α-HBD),
is outlined in Equation 13-8: Aspartate Aminotransferase
Aspartate aminotransferase (AST) is an enzyme belong-
CH3 CH3 ing to the class of transferases. It is commonly referred
 -HBD
 to as a transaminase and is involved in the transfer of an
CH2 NADH H CH2 NAD amino group between aspartate and α-keto acids. The
  older terminology, serum glutamic oxaloacetic transami-
HCBO HCMOH nase (SGOT, or GOT), may also be used. Pyridoxal phos-
  phate functions as a coenzyme. The reaction proceeds
COOH COOH according to Equation 12-10:
-Ketobutyrate -Hydroxybutyrate
(Eq. 13-8) COOH COOH COOH COOH
  AST
 
α-HBD is not a separate and distinct enzyme but is CH2 CH2 CH2 CH2
considered to represent the LD-1 activity of total LD.    
However, α-HBD activity is not entirely specific for HCM NH2 CH2 CBO CH2
the LD-1 fraction because LD-2, LD-3, and LD-4 also
   
contain varying amounts of the H subunit. HBD activity
COOH CBO COOH HCMNH2
is increased in those conditions in which the LD-1 and
 
LD-2 fractions are increased.
COOH COOH
LD is commonly used to measure lactic and pyruvic
Asparate -Keto- Oxalo- Glutamate
acids or as a coupled reaction.
glutarate acetate
Assay for Enzyme Activity (Eq. 13-10)
LD catalyzes the interconversion of lactic and pyruvic
acids using the coenzyme NAD+. The reaction sequence The transamination reaction is important in interme-
is outlined in Equation 13-9: diary metabolism because of its function in the synthesis
and degradation of amino acids. The ketoacids formed by
Lactate + NAD+ ∆ Pyruavate + NADH + H+ the reaction are ultimately oxidized by the tricarboxylic
(Eq. 13-9) acid cycle to provide a source of energy.

The reaction can proceed in either a forward (lactate Tissue Source

[L]) or reverse (pyruvate [P]) direction. Both reactions AST is widely distributed in human tissue. The highest
have been used in clinical assays. The rate of the reverse concentrations are found in cardiac tissue, liver, and
reaction is approximately three times faster, allowing skeletal muscle, with smaller amounts found in the kid-
smaller sample volumes and shorter reaction times. ney, pancreas, and erythrocytes.
278 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

Diagnostic Significance
The clinical use of AST is limited mainly to the evalu- CASE STUDY 13-2
ation of hepatocellular disorders and skeletal muscle While a 71-year-old woman is walking home from
involvement. In AMI, AST levels begin to rise within 6 to a shopping center, she faints and falls. She is driven
8 hours, peak at 24 hours, and generally return to normal home by a friend. When home, she realizes that
within 5 days. However, because of the wide tissue distri- she is bleeding from her mouth and is slightly
bution, AST levels are not useful in the diagnosis of AMI. disoriented. She appears injured from the fall, but
AST elevations are frequently seen in pulmonary she does not remember tripping or falling. The
embolism. Following congestive heart failure, AST levels woman is taken to a local emergency department.
also may be increased, probably reflecting liver involve- The examining physician determines that there was
ment as a result of inadequate blood supply to that organ. a loss of consciousness; to determine the reason,
AST levels are highest in acute hepatocellular disorders. he orders a head CT and ECG and the following
In viral hepatitis, levels may reach 100 times the ULN. laboratory tests: CBC, PT, aPTT, CK, LD, AST,
In cirrhosis, only moderate levels—approximately four and troponin T and troponin I. All tests are within
times the ULN—are detected (see Chapter 25). Skeletal normal limits. The woman is sutured for the mouth
muscle disorders, such as the muscular dystrophies, and injuries and admitted to a 24-hour observation unit.
inflammatory conditions also cause increases in AST
levels (4 to 8× ULN). Questions
AST exists as two isoenzyme fractions located in the
cell cytoplasm and mitochondria. The intracellular con- 1. What possible diagnoses is the physician
centration of AST may be 7,000 times higher than the considering?
extracellular concentration. The cytoplasmic isoenzyme 2. What laboratory tests would be elevated at 6, 12,
is the predominant form occurring in serum. In disorders and 24 hours if this patient had an AMI?
producing cellular necrosis, the mitochondrial form may
be significantly increased. Isoenzyme analysis of AST is 3. What isoenzyme tests would be useful with this
not routinely performed in the clinical laboratory. patient?

Assay for Enzyme Activity

Assay methods for AST are generally based on the pyruvic transaminase (SGPT, or GPT). Equation 13-12
principle of the Karmen method, which incorporates a indicates the transferase reaction. Pyridoxal phosphate
coupled enzymatic reaction using malate dehydrogenase acts as the coenzyme:
as the indicator reaction and monitors the change in
absorbance at 340 nm continuously as NADH is oxidized CH3 COOH CH3 COOH
to NAD+ (Equation 13-11). The optimal pH is 7.3 to 7.8:
ALT
AST
HCM NH2 CBO CBO HCM NH2
Aspartate + α-ketoglutarate ∆
Oxaloacetate + glutamate COOH CH2 COOH CH2
Oxaloacetate + NADH + H+ ∆ Malate + NAD+
(Eq. 13-11) CH2 CH2

Source of Error COOH COOH

Hemolysis should be avoided because it can dramatically Alanine -Keto- Pyruvate Glutamate
increase serum AST concentration. AST activity is stable glutarate
in serum for 3 to 4 days at refrigerated temperatures.
(Eq. 13-12)
Reference Range
AST, 5 to 35 U/L (37°C) (0.1 to 0.6 µkat/L) Tissue Source
ALT is distributed in many tissues, with comparatively
Alanine Aminotransferase high concentrations in the liver. It is considered the
more liver-specific enzyme of the transferases.
Alanine aminotransferase (ALT) is a transferase with
enzymatic activity similar to that of AST. Specifically, Diagnostic Significance
it catalyzes the transfer of an amino group from alanine Clinical applications of ALT assays are confined mainly
to α-ketoglutarate with the formation of glutamate and to evaluation of hepatic disorders. Higher elevations are
pyruvate. The older terminology was serum glutamic found in hepatocellular disorders than in extrahepatic or
 CHAPTER 13 n ENZYMES 279

intrahepatic obstructive disorders. In acute inflammatory Tissue Source

conditions of the liver, ALT elevations are frequently ALP activity is present on cell surfaces in most human
higher than those of AST and tend to remain elevated tissue. The highest concentrations are found in the
longer as a result of the longer half-life of ALT in serum intestine, liver, bone, spleen, placenta, and kidney. In the
(16 and 24 hours, respectively). liver, the enzyme is located on both sinusoidal and bile
Cardiac tissue contains a small amount of ALT activ- canalicular membranes; activity in bone is confined to
ity, but the serum level usually remains normal in AMI the osteoblasts, those cells involved in the production of
unless subsequent liver damage has occurred. ALT levels bone matrix. The specific location of the enzyme within
have historically been compared with levels of AST to this tissue accounts for the more predominant elevations
help determine the source of an elevated AST level and in certain disorders.
to detect liver involvement concurrent with myocardial
injury. Diagnostic Significance
Elevations of ALP are of most diagnostic significance in
Assay for Enzyme Activity the evaluation of hepatobiliary and bone disorders. In
The typical assay procedure for ALT consists of a hepatobiliary disorders, elevations are more predomi-
coupled enzymatic reaction using LD as the indicator nant in obstructive conditions than in hepatocellular dis-
enzyme, which catalyzes the reduction of pyruvate to orders; in bone disorders, elevations are observed when
lactate with the simultaneous oxidation of NADH. The there is involvement of osteoblasts.
change in absorbance at 340 nm measured continuously In biliary tract obstruction, ALP levels range from
is directly proportional to ALT activity. The reaction 3 to 10 times the ULN. Increases are primarily a result
proceeds according to Equation 13-13. The optimal pH of increased synthesis of the enzyme induced by cho-
is 7.3 to 7.8: lestasis. In contrast, hepatocellular disorders, such as
ALT
Alanine + α-ketoglutarate ∆ Pyruvate + glutamate hepatitis and cirrhosis, show only slight increases, usu-
LD
Pyruvate + NADH + H+ ∆ Lactate + NAD+ ally less than three times the ULN. Because of the degree
(Eq. 13-13)
of overlap of ALP elevations that occurs in the various
liver disorders, a single elevated ALP level is difficult to
Source of Error interpret. It assumes more diagnostic significance when
ALT is stable for 3 to 4 days at 4°C. It is relatively unaf- evaluated along with other tests of hepatic function (see
fected by hemolysis. Chapter 25).
Elevated ALP levels may be observed in various bone
Reference Range disorders. Perhaps the highest elevations of ALP activity
ALT, 7 to 45 U/L (37°C) (0.1 to 0.8 µkat/L) occur in Paget’s disease (osteitis deformans). Other bone
disorders include osteomalacia, rickets, hyperparathy-
Alkaline Phosphatase roidism, and osteogenic sarcoma. In addition, increased
ALP belongs to a group of enzymes that catalyze the levels are observed in healing bone fractures and during
hydrolysis of various phosphomonoesters at an alkaline periods of physiologic bone growth.
pH. Consequently, ALP is a nonspecific enzyme capable In normal pregnancy, increased ALP activity, averag-
of reacting with many different substrates. Specifically, ing approximately 1½ times the ULN, can be detected
ALP functions to liberate inorganic phosphate from an between weeks 16 and 20 and is two to three times the
organic phosphate ester with the concomitant produc- ULN during the third trimester. ALP activity increases
tion of an alcohol. The reaction proceeds according to and persists until the onset of labor. Activity then returns
Equation 13-14: to normal within 3 to 6 days.24 Elevations also may be
seen in complications of pregnancy such as hyperten-
O O sion, preeclampsia, and eclampsia, as well as in threat-
 ALP
 ened abortion.
RMPMO H2O RMOH HOMPMO ALP levels are significantly decreased in the inherited
pH 9-10
  condition of hypophosphatasia. Subnormal activity is a
O O result of the absence of the bone isoenzyme and results
Phosophomonoester Alcohol Phosphate ion in inadequate bone calcification.
(Eq. 13-14) ALP exists as a number of isoenzymes, which have
been studied by a variety of techniques. The major iso-
The optimal pH for the reaction is 9.0 to 10.0, but enzymes, which are found in the serum and have been
optimal pH varies with the substrate used. The enzyme most extensively studied, are those derived from the
requires Mg2+ as an activator. liver, bone, intestine, and placenta.25
280 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

Electrophoresis is considered the most useful sin- of the four major fractions, followed by intestinal, liver,
gle technique for ALP isoenzyme analysis. However, and bone fractions in decreasing order of heat stability.
because there may still be some degree of overlap Placental ALP will resist heat denaturation at 65°C for
between the fractions, electrophoresis in combination 30 minutes.
with another separation technique may provide the most Heat inactivation is an imprecise method for dif-
reliable information. A direct immunochemical method ferentiation because inactivation depends on many
for the measurement of bone-related ALP is now avail- factors, such as correct temperature control, timing,
able; this has made ALP electrophoresis unnecessary in and analytic methods sensitive enough to detect small
most cases. amounts of residual ALP activity. In addition, there
The liver fraction migrates the fastest, followed by is some degree of overlap between heat inactivation
bone, placental, and intestinal fractions. Because of of liver and bone fractions in both liver and bone
the similarity between liver and bone phosphatases, diseases.
there often is not a clear separation between them. A third approach to identification of ALP isoenzymes
Quantitation with use of a densitometer is sometimes is based on selective chemical inhibition. Phenylalanine
difficult because of the overlap between the two peaks. is one of several inhibitors that have been used.
The liver isoenzyme can actually be divided into two Phenylalanine inhibits intestinal and placental ALP to
fractions—the major liver band and a smaller fraction a much greater extent than liver and bone ALP. With
called fast liver, or α1 liver, which migrates anodal phenylalanine use, however, it is impossible to dif-
to the major band and corresponds to the α1 fraction ferentiate placental from intestinal ALP or liver from
of protein electrophoresis. When total ALP levels are bone ALP.
increased, the major liver fraction is the most fre- In addition to the four major ALP isoenzyme frac-
quently elevated. Many hepatobiliary conditions cause tions, certain abnormal fractions are associated with
elevations of this fraction, usually early in the course neoplasms. The most frequently seen are the Regan
of the disease. The fast-liver fraction has been reported and Nagao isoenzymes. They have been referred to as
in metastatic carcinoma of the liver, as well as in other carcinoplacental ALPs because of their similarities to
hepatobiliary diseases. Its presence is regarded as a the placental isoenzyme. The frequency of occurrence
valuable indicator of obstructive liver disease. However, ranges from 3% to 15% in cancer patients. The Regan
it is occasionally present in the absence of any detect- isoenzyme has been characterized as an example of an
able disease state. ectopic production of an enzyme by malignant tissue. It
The bone isoenzyme increases due to osteoblastic has been detected in various carcinomas, such as lung,
activity and is normally elevated in children during breast, ovarian, and colon, with the highest incidences
periods of growth and in adults older than age 50. In in ovarian and gynecologic cancers. Because of its low
these cases, an elevated ALP level may be difficult to incidence in cancer patients, diagnosis of malignancy
interpret.26 is rarely based on its presence. It is, however, useful in
The presence of intestinal ALP isoenzyme in serum monitoring the effects of therapy because it will disap-
depends on the blood group and secretor status of the pear on successful treatment.
individual. Individuals who have B or O blood group The Regan isoenzyme migrates to the same position as
and are secretors are more likely to have this fraction. the bone fraction and is the most heat stable of all ALP
Apparently, intestinal ALP is bound by erythrocytes of isoenzymes, resisting denaturation at 65°C for 30 min-
group A. Furthermore, in these individuals, increases in utes. Its activity is inhibited by phenylalanine.
intestinal ALP occur after consumption of a fatty meal. The Nagao isoenzyme may be considered a variant of
Intestinal ALP may increase in several disorders, such the Regan isoenzyme. Its electrophoretic, heat stability,
as diseases of the digestive tract and cirrhosis. Increased and phenylalanine inhibition properties are identical to
levels are also found in patients undergoing chronic those of the Regan fraction. However, Nagao also can be
hemodialysis. inhibited by L-leucine. Its presence has been detected in
Difference in heat stability is the basis of a second metastatic carcinoma of pleural surfaces and in adeno-
approach used to identify the isoenzyme source of an carcinoma of the pancreas and bile duct.
elevated ALP. Typically, ALP activity is measured before
and after heating the serum at 56°C for 10 minutes. If Assay for Enzyme Activity
the residual activity after heating is less than 20% of the Because of the relative nonspecificity of ALP with
total activity before heating, then the ALP elevation is regard to substrates, a variety of methodologies for its
assumed to be a result of bone phosphatase. If greater analysis have been proposed and are still in use today.
than 20% of the activity remains, the elevation is prob- The major differences between these relate to the
ably a result of liver phosphatase. These results are based concentration and types of substrate and buffer used
on the finding that placental ALP is the most heat stable and the pH of the reaction. A continuous-monitoring
 CHAPTER 13 n ENZYMES 281

technique based on a method devised by Bowers and O O

McComb allows calculation of ALP activity based on  
ACP
the molar absorptivity of p-nitrophenol. The reaction RMP MO H2O pH 5
RMOH HOMPMO
proceeds according to Equation 13-15:  
O O
O O O O Phosphomonoester Alcohol Phosphate ion
B

B
B

B
(Eq. 13-16)
N N
M

M
Tissue Source
ALP
! HO M P M O– ACP activity is found in the prostate, bone, liver, spleen,
pH 10.2 kidney, erythrocytes, and platelets. The prostate is the
richest source, with many times the activity found in
M

O O– other tissue.
M

Diagnostic Significance
HO M P M O–
Historically, ACP measurement has been used as an
B

O aid in the detection of prostatic carcinoma, particularly

metastatic carcinoma of the prostate. Total ACP deter-
p-Nitrophenyl- p-Nitro- Phosphate ion minations are relatively insensitive techniques, detecting
phosphate phenol elevated ACP levels resulting from prostatic carcinoma
(Eq. 13-15) in the majority of cases only when the tumor has metas-
tasized. Newer markers, such as prostate-specific antigen
p-Nitrophenylphosphate (colorless) is hydrolyzed to (PSA), are more useful screening and diagnostic tools
p-nitrophenol (yellow), and the increase in absorbance (see Chapter 34).
at 405 nm, which is directly proportional to ALP activity, One of the most specific substrates for prostatic ACP
is measured. is thymolphthalein monophosphate. Chemical inhibition
methods used to differentiate the prostatic portion most
Source of Error frequently use tartrate as the inhibitor. The prostatic
Hemolysis may cause slight elevations because ALP is fraction is inhibited by tartrate. Serum and substrate
approximately six times more concentrated in erythro- are incubated both with and without the addition of
cytes than in serum. ALP assays should be run as soon L-tartrate. ACP activity remaining after inhibition with
as possible after collection. Activity in serum increases L-tartrate is subtracted from the total ACP activity deter-
approximately 3% to 10% on standing at 25°C or 4°C for mined without inhibition, and the difference represents
several hours. Diet may induce elevations in ALP activity the prostatic portion:
of blood group B and O individuals who are secretors.
Total ACP − ACP after tartrate inhibition
Values may be 25% higher following ingestion of a high-
= Prostatic ACP (Eq. 13-17)
fat meal.
The reaction is not entirely specific for prostatic ACP;
Reference Range lysosomal ACP is also inhibited by tartrate. However,
ALP (total) (37°C) other tissue sources are largely uninhibited.
Males/Females 4–15 y 54–369 U/L (0.9–6.3 µkat/L) Neither of these methods of ACP determination is
sensitive to prostatic carcinoma that has not metasta-
Males 20–50 y 53–128 U/L (0.9–2.1 µkat/L) sized. Values are usually normal in the majority of cases
≥60 y 56–119 U/L (0.9–2.0 µkat/L) and, in fact, may be elevated only in about 50% of cases
Females 20–50 y 42–98 U/L (0.7–1.6 µkat/L) of prostatic carcinoma that has metastasized.
≥60 y 53–141 U/L (0.9–2.4 µkat/L) One technique with much improved sensitivity over
conventional ACP assays is the immunologic approach
using antibodies that are specific for the prostatic por-
Acid Phosphatase
tion. Immunochemical techniques, however, are not of
Acid phosphatase (ACP) belongs to the same group of value as screening tests for prostatic carcinoma.
phosphatase enzymes as ALP and is a hydrolase that PSA is more likely than ACP to be elevated at each
catalyzes the same type of reactions. The major differ- stage of prostatic carcinoma, even though a normal PSA
ence between ACP and ALP is the pH of the reaction. level may be found in stage D tumors. PSA is particularly
ACP functions at an optimal pH of approximately 5.0. useful to monitor the success of treatment; however,
Equation 13-16 outlines the reaction sequence: PSA is controversial as a screening test for prostatic
282 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

malignancy because PSA elevation may occur in condi- hours if the sample is left at room temperature without
tions other than prostatic carcinoma, such as benign the addition of a preservative. Decreased activity is a
prostatic hypertrophy and prostatitis.27-29 result of a loss of carbon dioxide from the serum, with
Other prostatic conditions in which ACP elevations a resultant increase in pH. If not assayed immediately,
have been reported include hyperplasia of the prostate serum should be frozen or acidified to a pH lower than
and prostatic surgery. There are conflicting reports of 6.5. With acidification, ACP is stable for 2 days at room
elevations following rectal examination and prostate temperature. Hemolysis should be avoided because of
massage. Certain studies have reported ACP elevations; contamination from erythrocyte ACP.
others have indicated no detectable change. When eleva- RIA procedures for measurement of prostatic ACP
tions are found, levels usually return to normal within require nonacidified serum samples. Activity is stable for
24 hours.30 2 days at 4°C.
ACP assays have proved useful in forensic clini-
cal chemistry, particularly in the investigation of rape. Reference Range
Vaginal washings are examined for seminal fluid–ACP Prostatic ACP, 0 to 3.5 ng/mL
activity, which can persist for up to 4 days.31 Elevated Tartrate-resistant ACP, adults: 1.5–4.5 U/L (37°C);
activity is presumptive evidence of rape in such cases. children: 3.5–9.0 U/L (37°C)
Serum ACP activity may frequently be elevated in
bone disease. Activity has been shown to be associated f-Glutamyltransferase
with the osteoclasts.32 Elevations have been noted in
γ-Glutamyltransferase (GGT) is an enzyme involved in
Paget’s disease, in breast cancer with bone metastases,
the transfer of the γ-glutamyl residue from γ-glutamyl
and in Gaucher’s disease, in which there is an infiltration
peptides to amino acids, H2O, and other small peptides.
of bone marrow and other tissue by Gaucher cells rich in
In most biologic systems, glutathione serves as the
ACP activity. Because of ACP activity in platelets, eleva-
γ-glutamyl donor. Equation 13-19 outlines the reaction
tions are observed when platelet damage occurs, as in
sequence:
the thrombocytopenia resulting from excessive platelet
AST
destruction from idiopathic thrombocytopenic purpura. Glutathione + amino acid ∆
Glutamyl peptide + L-cysteinylglycine
Assay for Enzyme Activity
Assay procedures for total ACP use the same techniques (Eq. 13-19)
as in ALP assays but are performed at an acid pH:
ACP
The specific physiologic function of GGT has not
p-Nitrophenolphosphate ∆pH 5 been clearly established, but it is suggested that GGT
p-Nitrophenol + phosphate ion (Eq. 13-18) is involved in peptide and protein synthesis, regulation
of tissue glutathione levels, and the transport of amino
The reaction products are colorless at the acid pH of acids across cell membranes.33
the reaction, but the addition of alkali stops the reaction
and transforms the products into chromogens, which can Tissue Source
be measured spectrophotometrically. GGT activity is found primarily in tissues of the kidney,
Some substrate specificities and chemical inhibitors brain, prostate, pancreas, and liver. Clinical applications
for prostatic ACP measurements have been discussed of assay, however, are confined mainly to evaluation of
previously. Thymolphthalein monophosphate is the sub- liver and biliary system disorders.
strate of choice for quantitative endpoint reactions. For
continuous-monitoring methods, α-naphthyl phosphate Diagnostic Significance
In the liver, GGT is located in the canaliculi of the
is preferred.
hepatic cells and particularly in the epithelial cells lining
Immunochemical techniques for prostatic ACP use
the biliary ductules. Because of these locations, GGT is
several approaches, including RIA, counterimmunoelec-
elevated in virtually all hepatobiliary disorders, making it
trophoresis, and immunoprecipitation. Also, an immu-
one of the most sensitive of enzyme assays in these con-
noenzymatic assay (Tandem E) includes incubation with
ditions (see Chapter 25). Higher elevations are generally
an antibody to prostatic ACP followed by washing and
observed in biliary tract obstruction.
incubation with p-nitrophenylphosphate. The p-nitro-
Within the hepatic parenchyma, GGT exists to a
phenol formed, measured photometrically, is propor-
large extent in the smooth endoplasmic reticulum and
tional to the prostatic ACP in the sample.
is, therefore, subject to hepatic microsomal induction.
Source of Error Therefore, GGT levels will be increased in patients
Serum should be separated from the red cells as soon as receiving enzyme-inducing drugs such as warfarin, phe-
the blood has clotted to prevent leakage of erythrocyte nobarbital, and phenytoin. Enzyme elevations may reach
and platelet ACP. Serum activity decreases within 1 to 2 levels four times the ULN.
 CHAPTER 13 n ENZYMES 283

Because of the effects of alcohol on GGT activity, Reference Range

elevated GGT levels may indicate alcoholism, particu- GGT: male, 6 to 55 U/L (37°C) (0.1 to 0.9 µkat/L);
larly chronic alcoholism. Generally, enzyme elevations female, 5 to 38 U/L (37°C) (0.1 to 0.6 µkat/L)
in persons who are alcoholics or heavy drinkers range Values are lower in females, presumably because of
from two to three times the ULN, although higher levels suppression of enzyme activity resulting from estrogenic
have been observed. GGT assays are useful in monitor- or progestational hormones.
ing the effects of abstention from alcohol and are used as
such by alcohol treatment centers. Levels usually return Amylase
to normal within 2 to 3 weeks after cessation but can rise
again if alcohol consumption is resumed. Because of the Amylase (AMY) is an enzyme belonging to the class
susceptibility to enzyme induction, any interpretation of hydrolases that catalyze the breakdown of starch
of GGT levels must be done with consideration of the and glycogen. Starch consists of both amylose and
consequent effects of drugs and alcohol. amylopectin. Amylose is a long, unbranched chain of
GGT levels are also elevated in other conditions, glucose molecules, linked by α, 1-4 glycosidic bonds;
such as acute pancreatitis, diabetes mellitus, and MI. amylopectin is a branched-chain polysaccharide with
The source of elevation in pancreatitis and diabetes is α, 1-6 linkages at the branch points. The structure of
probably the pancreas, but the source of GGT in MI is glycogen is similar to that of amylopectin but is more
unknown. GGT assays are of limited value in the diag- highly branched. α-AMY attacks only the α, 1-4 glyco-
nosis of these conditions and are not routinely requested. sidic bonds to produce degradation products consisting
GGT activity is useful in differentiating the source of of glucose; maltose; and intermediate chains, called
an elevated ALP level because GGT levels are normal in dextrins, which contain α, 1-6 branching linkages.
skeletal disorders and during pregnancy. It is particularly Cellulose and other structural polysaccharides con-
useful in evaluating hepatobiliary involvement in adoles- sisting of linkages are not attacked by α-AMY. AMY
cents because ALP activity will invariably be elevated as is therefore an important enzyme in the physiologic
a result of bone growth. digestion of starches. The reaction proceeds according
to Equation 13-21:
Assay for Enzyme Activity
The most widely accepted substrate for use in GGT anal- CH2OH CH2OH CH2OH
O H O H O H
M

M
ysis is γ-glutamyl-p-nitroanilide. The γ-glutamyl residue glucose,
AMS
M

M
is transferred to glycylglycine, releasing p-nitroaniline, OH– M O M OH M O M OH M O... maltose,
HO dextrins
M
M

M
M

M
M
a chromogenic product with a strong absorbance at 405
to 420 nm. The reaction, which can be used as a contin- OH OH OH
uous-monitoring or fixed-point method, is outlined in (Eq. 13-21)
Equation 13-20:
AMY requires calcium and chloride ions for its
HOOC M CHNH2 M CH2 M CH2 M CO activation.
M

HN M M NO2 Tissue Source

γ-Glutamyl-p-nitroanilide The acinar cells of the pancreas and the salivary glands
! H2N M CH2 M CONH M CH2 M COOH are the major tissue sources of serum AMY. Lesser
Glycylglycine concentrations are found in skeletal muscle and the
HOOC M CHNH2 M CH2 M CH2 M CO
small intestine and fallopian tubes. AMY is the smallest
enzyme, with a molecular weight of 50,000 to 55,000 Da.
M

HN M CH2 M CONH M CH2 M COOH Because of its small size, it is readily filtered by the renal
γ-Glutamyl-glycylglycine glomerulus and also appears in the urine.
GGT
! Digestion of starches begins in the mouth with the
hydrolytic action of salivary AMY. Salivary AMY activity,
pH 8.2 H2N M M NO2
however, is of short duration because, on swallowing,
p-Nitroaniline it is inactivated by the acidity of the gastric contents.
(Eq. 13-20) Pancreatic AMY then performs the major digestive action
of starches once the polysaccharides reach the intestine.

Source of Error Diagnostic Significance

GGT activity is stable, with no loss of activity for 1 week The diagnostic significance of serum and urine AMY
at 4°C. Hemolysis does not interfere with GGT levels measurements is in the diagnosis of acute pancreati-
because the enzyme is lacking in erythrocytes. tis.34 Disorders of tissue other than the pancreas can
284 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

also produce elevations in AMY levels. Therefore, electrophoresis. The most commonly observed fractions
an elevated AMY level is a nonspecific finding. are P2, S1, and S2.
However, the degree of elevation of AMY is helpful, In acute pancreatitis, there is typically an increase
to some extent, in the differential diagnosis of acute in P-type activity, with P3 being the most predomi-
pancreatitis. In addition, other laboratory tests (e.g., nant isoenzyme. However, P3 also has been detected
measurements of urinary AMY levels, AMY clearance in cases of renal failure and, therefore, is not entirely
studies, AMY isoenzyme studies, and measurements specific for acute pancreatitis. S-type isoamylase rep-
of serum lipase [LPS] levels), when used in conjunc- resents approximately two-thirds of AMY activity of
tion with serum AMY measurement, increase the normal serum, whereas P-type predominates in normal
specificity of AMY measurements in the diagnosis of urine.
acute pancreatitis.
In acute pancreatitis, serum AMY levels begin to rise 5 Assay for Enzyme Activity
AMY can be assayed by a variety of different methods,
to 8 hours after the onset of an attack, peak at 24 h, and
which are summarized in Table 13-6. The four main
return to normal levels within 3 to 5 days. Values gener-
approaches are categorized as amyloclast, saccharogenic,
ally range from 250 to 1,000 Somogyi units per dL (2.55×
chromogenic, and continuous monitoring. Continuous
ULN). Values can reach much higher levels.
monitoring has replaced previous methods for AMY
Other disorders causing an elevated serum AMY
activity.
level include salivary gland lesions, such as mumps and
In the amyloclastic method, AMY is allowed to act on
parotitis, and other intra-abdominal diseases, such as
perforated peptic ulcer, intestinal obstruction, cholecys- a starch substrate to which iodine has been attached. As
AMY hydrolyzes the starch molecule into smaller units,
titis, ruptured ectopic pregnancy, mesenteric infarction,
the iodine is released and a decrease occurs in the initial
and acute appendicitis. In addition, elevations have been
dark-blue color intensity of the starch–iodine complex.
reported in renal insufficiency and diabetic ketoacidosis.
The decrease in color is proportional to the AMY con-
Serum AMY levels in intra-abdominal conditions other
centration.
than acute pancreatitis are usually less than 500 Somogyi
The saccharogenic method uses a starch substrate that
units per dL.
is hydrolyzed by the action of AMY to its constituent
An apparently asymptomatic condition of hyperamy-
carbohydrate molecules that have reducing properties.
lasemia has been noted in approximately 1% to 2% of the
The amount of reducing sugars is then measured where
population. Hyperamylasemia can occur in neoplastic
the concentration is proportional to AMY activity. The
diseases with elevated results as high as 50 times the
saccharogenic method, the classic reference method for
ULN. Macroamylasemia is a condition that results when
determining AMY activity, is reported in Somogyi units.
the AMY molecule combines with immunoglobulins to
Somogyi units are an expression of the number of mil-
form a complex that is too large to be filtered across the
ligrams of glucose released in 30 minutes at 37°C under
glomerulus. Serum AMY levels increase because of the
specific assay conditions.
reduction in normal renal clearance of the enzyme, and
Chromogenic methods use a starch substrate to
consequently, the urinary excretion of AMY is abnormal-
which a chromogenic dye has been attached, forming an
ly low. The diagnostic significance of macroamylasemia
insoluble dye–substrate complex. As AMY hydrolyzes
lies in the need to differentiate it from other causes of
the starch substrate, smaller dye–substrate fragments are
hyperamylasemia.
produced, and these are water soluble. The increase in
Much interest has been focused recently on the possi-
ble diagnostic use of AMY isoenzyme measurements.34,35
Serum AMY is a mixture of a number of isoenzymes that
can be separated on the basis of differences in physi-
cal properties, most notably electrophoresis, although TABLE 13-6 AMYLASE METHODOLOGIES
chromatography and isoelectric focusing also have been Amyloclastic Measures the disappearance of
applied. In normal human serum, two major bands and starch substrate
as many as four minor bands may be seen. The bands Saccharogenic Measures the appearance of the
are designated as P-type and S-type isoamylase. P iso- product
amylase is derived from pancreatic tissue; S isoamylase Chromogenic Measures the increasing color from
is derived from salivary gland tissue, as well as the fal- production of product coupled
lopian tube and lung. The isoenzymes of salivary origin with a chromogenic dye
(S1, S2, and S3) migrate most quickly, whereas those of Continuous Coupling of several enzyme
pancreatic origin (P1, P2, and P3) are slower. In normal monitoring systems to monitor amylase
human serum, the isoamylases migrate in regions cor- activity
responding to the β- to α-globulin regions of protein
 CHAPTER 13 n ENZYMES 285

color intensity of the soluble dye–substrate solution is O

proportional to AMY activity. ||
CH2 O C R1 CH2OH
Recently, coupled-enzyme systems have been used | |
to determine AMY activity by a continuous-monitoring | O | O
technique in which the change in absorbance of NAD+ | || LPS | ||
CH2 O C R2 2H2O CH O C R2 2 fatty
at 340 nm is measured. Equation 13-22 is an example of | | acids
a continuous-monitoring method. For AMY activity, the | O CH2OH
optimal pH is 6.9: | ||
CH2 O C R3
AMS
Maltopentose Maltrotriose maltose Triacylgycerol 2-Monoglyceride
-glucosidase (Eq. 13-23)
Maltrotriose maltose 5-Glucose
Hexokinase
5-Glucose 5 ATP The enzymatic activity of pancreatic LPS is specific for
5-Glucose-6-phosphate 5 ADP the fatty acid residues at positions 1 and 3 of the triglyc-
G-6-PD eride molecule, but substrate must be an emulsion for
5-Glucose-6-phosphate 5 NAD activity to occur. The reaction rate is accelerated by the
5,6-Phosphogluconolactone 5 NADH presence of colipase and a bile salt.
(Eq. 13-22)
Tissue Source
Because salivary AMY is preferentially inhibited by LPS concentration is found primarily in the pancreas,
wheat germ lectin, salivary and pancreatic AMY can be although it is also present in the stomach and small
estimated by measuring total AMY in the presence and intestine.
absence of lectin. Specific immunoassays are also avail-
able for measuring isoenzymes of AMY.
Diagnostic Significance
Clinical assays of serum LPS measurements are confined
Source of Error almost exclusively to the diagnosis of acute pancreatitis.
AMY in serum and urine is stable. Little loss of activ- Serum LPS activity increases 4 to 8 hours after an attack
ity occurs at room temperature for 1 week or at 4°C for of acute pancreatitis; concentrations peak at 24 hours
2 months. Because plasma triglycerides suppress or and decrease within 8 to 14 days. LPS is similar in this
inhibit serum AMY activity, AMY values may be normal respect to AMY measurements but is considered more
in acute pancreatitis with hyperlipemia. specific for pancreatic disorders than AMY measurement.
The administration of morphine and other opiates Both AMY and LPS levels rise quickly, but LPS elevations
for pain relief before blood sampling will lead to falsely persist for approximately 8 days in acute pancreatitis,
elevated serum AMY levels. The drugs presumably cause whereas AMY elevations persist for only 2 to 3 days. The
constriction of the sphincter of Oddi and of the pan- extent of elevations does not correlate with severity of
creatic ducts, with consequent elevation of inarticulate disease. Elevated LPS levels also may be found in other
pressure causing regurgitation of AMY into the serum. intra-abdominal conditions but with less frequency than
elevations of serum AMY. Elevations have been reported
Reference Range in cases of penetrating duodenal ulcers and perforated
AMY: serum, 28 to 100 U/L (37°C) (0.5 to 1.7 µkat/L); peptic ulcers, intestinal obstruction, and acute cholecys-
urine, 1 to 15 U/h titis. In contrast to AMY levels, LPS levels are normal in
Because of the various AMY procedures currently conditions of salivary gland involvement. Therefore, LPS
in use, activity is expressed according to each proce- levels are useful in differentiating serum AMY elevation
dure. There is no uniform expression of AMY activ- as a result of pancreatic versus salivary involvement. Of
ity, although Somogyi units are frequently used. The the three LPS isoenzymes, L2 is thought to be the most
approximate conversion factor between Somogyi units clinically specific and sensitive.
and IUs is 1.85.
Assay for Enzyme Activity
Lipase Procedures used to measure LPS activity include estima-
tion of liberated fatty acids (titrimetric) and turbidimet-
LPS is an enzyme that hydrolyzes the ester linkages of ric methods. The reaction is outlined in Equation 13-24:
fats to produce alcohols and fatty acids. Specifically, LPS
catalyzes the partial hydrolysis of dietary triglycerides in LPS
Triglyceride 2 H2O
(pH 8.6–9.0)
the intestine to the 2-monoglyceride intermediate, with
the production of long-chain fatty acids. The reaction 2-Monoglyceride 2 fatty acids
proceeds according to Equation 13-23: (Eq. 13-24)
286 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

Early methods for LPS were historically poor. The Tissue Source
classic Cherry-Crandall method used an olive oil sub- Sources of G-6-PD include the adrenal cortex, spleen,
strate and measured the liberated fatty acids by titration thymus, lymph nodes, lactating mammary gland, and
after a 24-hour incubation. Modifications of the Cherry- erythrocytes. Little activity is found in normal serum.
Crandall method have been complicated by the lack of
stable and uniform substrates. However, triolein is one Diagnostic Significance
substrate now used as a more pure form of triglyceride. Most of the research interest in G-6-PD focuses on its
Turbidimetric methods are simpler and more rapid role in the erythrocyte. Here, it functions to maintain
than titrimetric assays. Fats in solution create a cloudy NADPH in reduced form. An adequate concentration of
emulsion. As the fats are hydrolyzed by LPS, the particles NADPH is required to regenerate sulfhydryl-containing
disperse, and the rate of clearing can be measured as an proteins, such as glutathione, from the oxidized to the
estimation of LPS activity. Colorimetric methods are reduced state. Glutathione in the reduced form, in turn,
also available and are based on coupled reactions with protects hemoglobin from oxidation by agents that may
enzymes such as peroxidase or glycerol kinase. be present in the cell. A deficiency of G-6-PD results in
an inadequate supply of NADPH and, ultimately, in the
Source of Error inability to maintain reduced glutathione levels. When
LPS is stable in serum, with negligible loss in activity erythrocytes are exposed to oxidizing agents, hemolysis
at room temperature for 1 week or for 3 weeks at 4°C. occurs because of oxidation of hemoglobin and damage
Hemolysis should be avoided because hemoglobin inhib- of the cell membrane.
its the activity of serum LPS, causing falsely low values. G-6-PD deficiency is an inherited sex-linked trait. The
disorder can result in several different clinical manifesta-
Reference Range
tions, one of which is drug-induced hemolytic anemia.
LPS, <38 U/L (37°C) (<0.6 µkat/L)
When exposed to an oxidant drug such as primaquine,
an antimalarial drug, affected individuals experience a
Glucose-6-Phosphate Dehydrogenase
hemolytic episode. The severity of the hemolysis is relat-
G-6-PD is an oxidoreductase that catalyzes the oxida- ed to the drug concentration. G-6-PD deficiency is most
tion of glucose-6-phosphate to 6-phosphogluconate or common in African Americans but has been reported in
the corresponding lactone. The reaction is important as virtually every ethnic group.
the first step in the pentose phosphate shunt of glucose Increased levels of G-6-PD in the serum have been
metabolism with the ultimate production of NADPH. reported in MI and megaloblastic anemias. No elevations
The reaction is outlined in Equation 13-25: are seen in hepatic disorders. G-6-PD levels, however,
are not routinely performed as diagnostic aids in these
OH
conditions.
|
HC |
| |
HC OH |
| |
G-6-PD
HO CH O NADP
| | CASE STUDY 13-3
HC OH |
| ||
HC A 36-year-old woman presents to the emergency
| department with intense upper abdominal pain radi-
CH2OPO3 ating to her back, weakness, loss of appetite, and
Glucose-6-phosphate severe indigestion after eating. She had not traveled
O in recent months. She has not been well for several
||
days.
C |
| |
HC OH | Questions
| |
HO CH O NADPH NH 1. What laboratory tests should be ordered to help
| | diagnose this patient?
HC OH |
| | 2. What enzyme tests will be useful in diagnosing
|
HC this patient?
|
CH2OPO3 3. What two diagnoses are most likely for this
6-Phosphogluconate patient?
(Eq. 13-25)
 CHAPTER 13 n ENZYMES 287

Assay for Enzyme Activity mal electrophoretic pattern of enzymes (see Fig. 13-5
The assay procedure for G-6-PD activity is outlined in for an example). Antienzyme antibodies can cause the
Equation 13-26: formation of new enzyme bands on a gel, can alter the
intensity of enzyme bands, and can cause band broaden-
G-6-PD
Glucose-6-phosphate NADPH ing on the gel.36 Other test methods used to determine
6-Phosphogluconate NADPH H the presence of macroenzymes include gel filtration,
(Eq. 13-26) immunoprecipitation, immunoelectrophoresis, counter-
immunoelectrophoresis, and immunofixation. Last, the
A red cell hemolysate is used to assay for deficiency immunoinhibition test can also be used to determine the
of the enzyme; serum is used for evaluation of enzyme presence of macro-CK.
elevations.
Drug-Metabolizing Enzymes
Reference Range
G-6-PD, 7.9 to 16.3 U/g Hgb (0.1 to 0.3 µkat/g Hgb) Drug-metabolizing enzymes function primarily to trans-
form xenobiotics into inactive, water-soluble compounds
Macroenzymes for excretion through the kidneys. Metabolic enzymes
Macroenzymes are high-molecular-mass forms of the can also transform inactive prodrugs into active drugs,
serum enzymes (ACP, ALP, ALT, AMY, AST, CK, GGT, convert xenobiotics into toxic compounds, or prolong
LD, and LPS) that can be bound to either an immuno- the elimination half-life. Drug-metabolizing enzymes cat-
globulin (macroenzyme type 1) or a nonimmunoglobu- alyze addition or removal of functional groups through
lin substance (macroenzyme type 2). Macroenzymes hydroxylation, oxidation, dealkylation, dehydrogena-
are usually found in patients who have an unexplained tion, reduction, deamination, and desulfuration reac-
persistent increase of enzyme concentrations in serum.36 tions. These transformation reactions are referred to as
The presence of macroenzymes can also increase with phase I reactions and are often mediated by cytochrome
increasing age.37 Enzymes can bind to immunoglobulins P450 (CYP 450) enzymes. Xenobiotics can also become
in a nonspecific manner, but there is also evidence that transformed into more polar compounds through
the enzyme–immunoglobulin complex can be formed by enzyme-mediated conjugation reactions, also known as
specific interactions between circulating autoantibodies phase II reactions, in which xenobiotics are conjugated
and serum enzymes.36 The reason for the formation of with glucuronide (UDP-glucuronosyltransferase 1A1
antienzyme antibodies is not known, but there are two [UGT1A1]), acetate (N-acetyltransferase [NAT]), gluta-
theories to explain their formation. According to the thione (glutathione-S-transferase [GST]), sulfate (sulfo-
“antigen-driven theory,” the self-antigen becomes immu- transferase), and methionine groups.39
nogenic by being altered or released from a sequestered CYP 450 enzymes are a superfamily of isoenzymes that
site and reacts with an antibody that is initially formed are involved in the metabolism of more than 50% of all
against a foreign antigen.38 The dysregulation of immune drugs. These enzymes contain heme molecules, and they
tolerance theory explains the formation of enzymes with are given the name CYP 450 because they absorb the max-
autoantibodies in patients with autoimmune disorders.38 imum amount of light at 450 nm. More than 500 CYP 450
To date, there has not been a strong correlation between enzymes have been identified, and they are classified into
the presence of antienzyme antibodies and the pathogen- families according to their homology to other enzymes.42
esis of disease.36 However, the presence of macroenzymes There are at least four CYP 450 (CYP1, 2, 3, and 4) fami-
should be documented in the patient’s medical records lies that are expressed primarily in the liver, but some iso-
because macroenzymes can persist for long periods.36 forms are also expressed in extrahepatic tissues such as the
Macroenzymes accumulate in plasma because their lung, kidney, gastrointestinal tract, skin, and placenta.39
high molecular masses prevent them from being filtered The specific isozyme is classified by not only its family
out of the plasma by the kidneys. The detection of mac- number but also a subfamily letter, a number for an indi-
roenzymes is clinically significant because the presence vidual isozyme within the subfamily, and, if applicable,
of macroenzymes can cause difficulty in the interpreta- an asterisk followed by a number for each genetic (allelic)
tion of diagnostic enzyme results. The formation of high- variant. Genetic variants have been identified that lead
molecular-weight enzyme complexes can cause false to complete enzyme deficiency (e.g., a frame shift, splice
elevations in plasma enzymes or they can falsely decrease variant, stop codon, or a complete gene deletion), reduced
the activity of the enzyme by blocking the activity of the enzyme function or expression, or enhanced enzyme
bound enzyme.36 function or expression. Recognition of genetic variants
The principal method to identify enzymes that are can explain interindividual differences in drug response
bound to immunoglobulins and nonimmunoglobulins and pharmacokinetics.40-43 For example, four phenotypes
is protein electrophoresis. The binding of enzymes to are recognized for CYP2D6: ultra-metabolizers, exten-
high-molecular-weight complexes can alter the nor- sive metabolizers, intermediate metabolizers, and poor
288 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

metabolizers. Patients who are poor metabolizers for the patients with nonfunctioning UGT1A1 are at risk for
CYP2D6 enzyme are at risk for therapeutic failure when hyperbilirubinema.43 Last, TPMT is an enzyme that can
inactive prodrugs such as tamoxifen require CYP2D6 for be found in bone marrow and erythrocytes and functions
drug activation. Tricyclic antidepressants such as nortrip- to inactivate chemotherapeutic thiopurine drugs like aza-
tyline require CYP2D6 for inactivation. Thus, CYP2D6 thioprine and 6-mercaptopurine. The TPMT enzyme has
poor metabolizers may require lower dose requirements genetic polymorphisms, which causes variable responses
than will patients with extensive (“normal”) metabolism (normal, intermediate, and low activity) to thiopurine
and may be at high risk for adverse drug reactions.39,41 metabolism. Patients with low TPMT activity are at risk
In addition to xenobiotic metabolism, CYP 450 enzymes for developing severe bone marrow toxicity when the
are also involved in the biosynthesis of endogenous standard dose therapy for thiopurine drugs is admin-
compounds. The CYP5 family consists of thromboxane istered; thus, genetic testing is essential for identifying
synthases that catalyze the reaction that leads to platelet patients with metabolizing enzyme polymorphisms.43
aggregation. CYP7 and CYP27 families catalyze the hydrox- Pharmacogenetic testing is often used prior to drug
ylation of cholesterol for the biosynthesis of bile acids. The therapy to assist clinicians in identifying patients with
CYP24 family catalyzes the hydroxylation and inactivation genetic polymorphisms and to guide drug and dose selec-
of vitamin D3. CYP 450 enzymes are also found in steroid- tion. Pharmacogenetic testing can be performed through
producing tissues and function to synthesize steroid hor- phenotype tests that measure metabolic enzyme activity,
mones from cholesterol (CYP11, 17, 19, and 21).42 through administration of a probe drug and subsequent
Genetic variants that affect drug-metabolizing enzyme evaluation of metabolic ratios, or through genotype test-
function and expression are recognized for other enzymes ing that identifies clinically significant genetic variants.
such as NAT, UGT1A1, GST, and thiopurine methyl- The activity of drug-metabolizing enzymes can also be
transferase (TPMT). These variants are associated with altered by food, nutritional supplements, or other drugs.
distinct extensive (fast), intermediate, or poor (slow) Compounds that stimulate an increase in the synthesis
metabolizer phenotypes, which could lead to adverse of CYP 450 enzymes are called inducers. Inducers will
drug reactions or therapeutic failure. For example, two increase the metabolism of drugs and reduce the bio-
phenotypes—fast or slow acetylators—are recognized availability of the parent compound. Compounds that
for N-acetyltransferase 2 (NAT2). NAT2 is the primary reduce the expression or activity of a drug-metabolizing
enzyme involved in the acetylation of isoniazid, a drug enzyme are referred to as inhibitors. For example, inhibi-
used to treat tuberculosis. Acetylation is the primary tors can compete with substrates for the active site of
mechanism for the elimination of isoniazid, and there- CYP 450 and thereby decrease the metabolism of drugs
fore, patients with low NAT2 activity will not be able to and increase the bioavailability of the parent compound,
inactivate isoniazid, putting those patients at increased or block activity or expression through noncompetitive
risk for adverse drug reactions.39 UGT1A1 has poly- means.39 Table 13-7 lists the common families of CYP
morphisms that can lead to a nonfunctioning enzyme. 450 enzymes along with some of their substrates and
UGT1A1 is responsible for the metabolism of bilirubin; drugs that can induce or inhibit enzyme activity.

TABLE 13-7 COMMON SUBSTRATES FOR DRUG-METABOLIZING ENZYMES

ENZYME SUBSTRATES INDUCERS INHIBITORS
CYP1A1 (R)-Warfarin Omeprazole
TCDD
Benzo[a]pyrene, 3MC
CYP1A2 Acetaminophen Insulin Ciprofloxacin
Caffeine Tobacco Cimetidine
(R)-Warfarin Polycyclic aromatic hydrocarbons Amiodarone
Estradiol Fluoroquinolones
Theophylline
CYP2A6 Cyclophosphamide Dexamethasone Pilocarpine
Halothane Coumarin
Zidovudine
Coumarin
(Continued)
 CHAPTER 13 n ENZYMES 289

TABLE 13-7 COMMON SUBSTRATES FOR DRUG-METABOLIZING ENZYMES (CONTINUED)

ENZYME SUBSTRATES INDUCERS INHIBITORS
CYP2B6 Cyclophosphamide Phenobarbital Ticlopidine
Diazepam Rifampin
Bupropion
CYP2C9 (S)-Warfarin Rifampin Fluconazole
Ibuprofen Secobarbital Amiodarone
Tolbutamide Isoniazid
Diclofenac Probenecid
Losartan Sertraline
Phenytoin Sulfamethoxazole
CYP2C19 Diazepam Barbiturate Omeprazole
Omeprazole Phenytoin Chloramphenicol
Chloripramine Cimetidine
Indomethacin Ketoconazole
Indomethacin
CYP2D6 Carvedilol Dexamethasone Bupropion
Amitriptyline Rifampin Fluoxetine
Haloperidol Quinidine
Amphetamine Amiodarone
Chlorpromazine Sertraline
Dextromethorphan Celecoxib
Codeine Chlorpromazine
CYP2E1 Acetaminophen Ethanol Disulfiram
Chlorzoxazone Isoniazid Diethyldithiocarbamate
Halothane
Ethanol
CYP3A4 Erythromycin HIV antivirals HIV antivirals
Quinidine Barbiturates Ketoconazole
Diazepam Carbamazepine Erythromycin
Cortisol Phenobarbital Grapefruit juice
Cyclosporine Phenytoin Cimetidine
Indinavir Rifampin Chloramphenicol
Chlorpheniramine St. John’s wort
Nifedipine Troglitazone
Lovastatin
Testosterone
Cocaine
Fentanyl
Tamoxifen
TPMT Azathioprine Naproxen
6-Mercaptopurine Furosemide

Source: Rendic S, Di Carlo FJ. Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors.
Drug Metab Rev. 1997;29:413-580.
290 PART 2 n CLINICAL CORRELATIONS AND ANALYTIC PROCEDURES

For additional student resources please visit thePoint at http://thepoint.lww.com.

QUESTIONS

1. When a reaction is performed in zero-order kinetics c. CK-MB

a. The rate of the reaction is independent of the d. CK-NN
substrate concentration
8. Elevation of serum amylase and lipase is commonly
b. The substrate concentration is very low
seen in
c. The rate of reaction is directly proportional to
a. Acute pancreatitis
the substrate concentration
b. Acute appendicitis
d. The enzyme level is always high
c. Gallbladder disease
2. Activation energy is d. Acid reflux disease
a. Decreased by enzymes
b. The energy needed for an enzyme reaction 9. The saccharogenic method for amylase determina-
to stop tions measures
c. Increased by enzymes a. The amount of product produced
d. Very high in catalyzed reactions b. The amount of substrate consumed
c. The amount of iodine present
3. Enzyme reaction rates are increased by increasing d. The amount of starch present
temperatures until they reach the point of denatur-
ation at 10. Elevation of tissue enzymes in serum may be used
a. 40–60°C to detect
b. 25–35°C a. Tissue necrosis or damage
c. 100°C b. Inflammation
d. 37°C c. Infectious diseases
d. Diabetes mellitus
4. An example of using enzymes as reagents in the
clinical laboratory is 11. Which of the following enzyme patterns is
a. The hexokinase glucose method MOST diagnostic of Duchenne-type muscular dys-
b. The diacetyl monoxime blood urea nitrogen trophy?
(BUN) method a. Total CK level that is 5 to 10 times the ULN
c. The alkaline picrate creatinine method b. Total CK level that is 25 times the ULN
d. The biuret total protein method c. Total CK level that is 50 to 100 times the ULN
d. Total CK level that is 1,000 times the ULN
5. Activity of enzymes in serum may be determined
rather than concentration because 12. Which of the following preanalytical errors most
a. The amount of enzyme is too low to measure commonly causes false increases in serum enzyme
b. The temperature is too high measurements?
c. There is not enough substrate a. The patient was not fasting prior to blood
d. The amount of enzyme is too high to measure draw.
b. The blood sample was not maintained on ice
6. The isoenzymes LD-4 and LD-5 are elevated in
upon collection and during transport to the
a. Liver disease
laboratory.
b. Pulmonary embolism
c. The serum was not separated from red blood
c. Renal disease
cells within 1 hour.
d. Myocardial infarction
d. The patient smoked three cigarettes just prior
7. Which CK isoenzyme is elevated in muscle to blood collection.
diseases? e. The blood sample was not protected from light
a. CK-MM upon collection and during transport to the
b. CK-BB laboratory.