128
chapter
8
Enzymes 111: Clinical Applications
hepatitis,
hemochromatosis,
Wilson’s
disease,
a\-
antitrypsin deficiency,
malignancy,
and poisons
and
infectious
agents.
These
disorders
require
specific
laboratory testing procedures and are discussed at the
appropriate places in the text. The serum enzymes used
in assessment of liver function are divided into two cat-
egories: (
1
) markers used in hepatocellular necrosis and
(2) markers that reflect cholestasis. Serum enzymes used
as markers of cholestasis include alkaline phosphatase,
5'-nucleotidase,
and
y
-glutamyl
transferase.
Alanine
aminotransferase and aspartate aminotransferases are
markers for hepatocellular necrosis. Other tests used in
the assessment of liver disorders include measurement of
bilirubin, albumin, and a-fetoprotein.
8.3 Enzymes as Analytical Reagents
The use of enzymes as analytical reagents in the clinical
laboratory has found widespread application in the mea-
surement of substrates, drugs, and activity of other en-
zymes. Above we discussed the use of enzymes as reagents
in the coupled assay of aspartate aminotransferase.
Enzyme-dependent procedures, used in the assay of sub-
strates such as glucose, urea, uric acid, and cholesterol,
provide many advantages over classical chemical proce-
dures. These advantages include specificity of the sub-
strate that is being measured and direct measurement of
the substrate in a complex mixture that avoids prelimi-
nary separation and purification steps such as serum. A
reagent enzyme can be used in substrate measurements by
two methods: end-point and rate assays. In an end-point
assay, the substrate is completely converted to product
before it is measured; in the rate assay, a change in sub-
strate concentration produced during a fixed-time interval
is measured. This second method, also referred to as the
two-point kinetic method, when carried out under con-
stant conditions of pH, temperature, amount of enzyme,
and time interval, yields very accurate values for substrate
concentration. This procedure is calibrated with standard
solutions of the substrate. Of course, the first-order reac-
tion condition with respect to substrate must be maintained
by keeping the reagent enzyme concentration high. Under
these conditions, when average velocities during the cho-
sen time interval are plotted against various standard sub-
strate concentrations, the profile obtained should exhibit a
straight line from which unknown values of substrate can
be calculated.
Optical properties can be used in monitoring assay pro-
cedures as they are in measuring enzyme activity. If the
primary substrate or product does not have a suitable
optical property (e.g., absorption in visible or ultravio-
let light), then a coupled assay is constructed in which one
or more auxiliary enzymes are employed to form an ul-
timate product that possesses an easily measured optical
property. For example, glucose in a biological specimen
can be measured by the following reactions under optimal
conditions:
hexokinase
D-Glucose
+ ATP-------
-> D-glucose 6-phosphate
+ ADP
gIucose-6-phosphatase
dehydrogenase
D-Glucose 6-phosphate
+ NADP+ <
±.
D-glucono-3-lactone 6-phosphate
+ NADPH + H+
The second reaction (in which NADPH is formed) func-
tions as the indicator reaction. Although the hexokinase
reaction is relatively nonspecific and acts on hexoses other
than glucose (Chapter 13) to form
6
-phosphate esters,
the indicator reaction is specific for glucose
6
-phosphate
making the overall assay procedure highly specific for
glucose.
Enzymes immobilized on an insoluble surface can be
used repeatedly in substrate assays. This type of assay is
feasible if the reaction product can be measured directly.
Immobilization can be accomplished through chemi-
cal linkages involving diazo, triazine, and azide groups
with many types of insoluble matrices, such as diethy-
laminoethylcellulose, carboxymethylcellulose, agarose,
microcrystalline cellulose, and inner walls of plastic tub-
ings. Enzymes immobilized on the inner surface of plastic
tubings are particularly useful in continuous-flow analyz-
ers. Reagent enzymes that have been immobilized include
glucose oxidase, urease, a-amylase, trypsin, and leucine
aminopeptidase.
Reagent
enzymes
and
antibodies
formed
against
specific molecules can be combined to determine the
concentration of a variety of molecules to which an-
tibodies (Chapter 35) can be formed. Such analytical
procedures are known as
enzyme immunoassays
(ElAs).
The reagent enzyme can be linked to antibodies or anti-
gens such that the complexes possess immunological or
enzymatic activity. Antibodies can be raised in verte-
brate animals when injected with specific proteins (anti-
gens) foreign to them. Macromolecules other than proteins
can also be antigenic. Low-molecular-weight compounds
(e.g., substrates, drugs) by themselves do not elicit an-
tibodies but do so if covalently linked to a carrier pro-
tein (e.g., albumin) before injection. The term
hapten
designates the low-molecular-weight substance that can
combine with the antibody produced against the carrier
protein complex. EIAs are either
heterogeneous
or
ho-
mogeneous.
A heterogeneous assay consists of at least
one separation step in which the bound enzyme-labeled
