section 8.1
Diagnosis and Prognosis of Disease
125
FIGURE 8-2
Diagrammatic representation of the measurement of the activity of an
enzyme, showing product formation as a function of time. The true activity
of the enzyme is calculated from data obtained when the reaction rate is
linear with maximum velocity. The reaction rate is directly proportional to
the amount of active enzyme only when the substrate concentrations are
maintained at saturating levels and when other variables (e.g., pH, and
cofactors) are held constant at optimal conditions.
enzyme, presence of certain endogenous metabolites, and
drugs (or their metabolites).
In the design of an enzyme assay, a key element is se-
lection of a suitable physical or chemical technique for
following the appearance of a product or the disappear-
ance of a substrate. The rate of enzymatic reactions can be
monitored continuously by photometric procedures (such
as light absorption, fluorescence, or optical rotation) if
the reaction is accompanied by a change in optical prop-
erties. For example, serum alkaline phosphatase, used in
the diagnosis of hepatobiliary and bone diseases, is mea-
sured by employing its ability to catalyze the hydrol-
ysis at alkaline pH of the colorless synthetic substrate
p-nitrophenylphosphate to the yellow-colored product
p-nitrophenol. The rate of reaction can be measured from
changes in absorption at 405 nm. Many enzymatic reac-
tions can be followed by a change in absorbance at 340 nm
if they are linked (either directly or indirectly) to a de-
hydrogenase reaction requiring a nicotinamide adenine-
dinucleotide (NAD+ or NADP+). The coenzyme absorbs
ultraviolet light at 340 nm in the reduced state but not in
the oxidized state (Figure 8-3). Hence, changes in reaction
rate in either direction can be followed by measurement
of the absorbance at 340 nm. An example of one such
enzyme assay is given below.
Red cell glucose-
6
-phosphate dehydrogenase (Chap-
ter 15) can be specifically assayed in a red cell hemolysate.
The enzyme catalyzes the reaction
D-Glucose
6
-phosphate + NADP+
D-glucono-5-lactone
6
-phosphate + NADPH + H+
FIGURE 8-3
Absorption spectra of NAD+ (NADP+) and NADH (NADPH). At 340 nm,
the reduced coenzymes (NADH or NADPH) show significant absorbance,
whereas the oxidized forms (NAD+ or NADP+) show negligible
absorbance. Thus, many enzymatic reactions can be monitored at 340 nm
if the reaction is directly or indirectly dependent upon a dehydrogenase
reaction involving a nicotinamide adenine dinucleotide as a coenzyme.
Since NADPH absorbs light at 340 nm and NADP+
does not (Figure 8-3), this reaction can be followed
by measurement of the change in absorbance at this
wavelength with time. A similar assay (with lactate and
NAD+) can be used for serum lactate dehydrogenase.
The difference in absorbance is measured so readily that
a number of assays make use of it directly or indi-
rectly. In the latter case, the reaction to be measured
is linked by one or more steps to a reaction in which
a nicotinamide adenine dinucleotide is oxidized or re-
duced. Thus, in the measurement of aminotransferases
(Chapter 17), one of the products of the primary reaction
serves as a substrate for a secondary reaction in which
NADH is required. For example, in the assay of aspartate
aminotransferase,
L-Aspartate +
a-ketoglutarate
«=2
oxaloacetate
(also known as 2-oxoglutarate)
+ L-glutamate (primary reaction)
the oxaloacetate formed in the primary reaction is reduced
to L-malate by
NADH
in the presence of malate dehydro-
genase:
malate
Oxaloacetate + NADH + H+------------
>
L-malate
dehydrogenase
+ NAD+ (indicator reaction)
This secondary reaction is an indicator reaction, i.e., the
one whose rate is followed. The primary, not the secondary,
