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chapter
8
Enzymes III: Clinical Applications
Presence
Removal
Normal turnover
of tissues
Leakage through
cell membranes
Tissue necrosis
increased enzyme
synthesis
intravascular inactivation
(dilution, lack of substrates and
coenzymes, inhibitors, and
proteinases)
Uptake by tissues, with
subsequent inactivation
Removal by the
reticuloendothelial system
Excretion in urine (of low-
molecular-weight enzymes)
F IG U R E 8-1
Factors affecting the presence and removal of non-plasma-specific
enzymes from plasma.
Inactivation or removal of plasma enzymes may be ac-
complished by several processes: dénaturation of the en-
zyme due to dilution in plasma or separation from its
natural substrate or coenzyme; presence of enzyme in-
hibitors (e.g., falsely decreased activity of amylase in acute
pancreatitis with hyperlipemia); removal by the reticu-
loendothelial system; digestion by circulating proteinases;
uptake by tissues and subsequent degradation by tissue
proteinases; and clearance by the kidneys of enzymes of
low molecular mass (amylase and lysozyme).
A schematic representation of the causes for the appear-
ance and disappearance of non-plasma-specific enzymes
is shown in Figure 8-1. Since enzymes differ in the rates of
their disappearance from plasma, it is important to know
when the blood specimen was obtained relative to the time
of injury. It is also important to know how soon after the oc-
currence of injury various enzyme levels begin to rise. The
biological half-lives for enzymes and their various isoen-
zymes are different. We can illustrate this by examining the
disappearance rates of the lactate dehydrogenase (LDH)
isoenzymes. LDH consists of four subunits of two different
types: H (heart) and M (muscle). The subunits combine to
yield a series of five tetramers: LDH](H4), LDH
2
(H
3
M),
LDH
3
(H
2
M2), LDH
4
(HM3), and LDH
5
(M4). The over-
all rate of disappearance of LDH activity from plasma
is biphasic because of the more rapid disappearance of
LDH
5
(the predominant isoenzyme of liver and skele-
tal muscle), followed by the relatively slower removal of
LDHi (the predominant isoenzyme of heart, kidney cor-
tex, and erythrocytes). The remaining LDH isoenzymes
have intermediate rates of disappearance. In chronic dis-
ease states, the plasma enzyme levels continue to be
elevated.
The use of appropriate normal ranges is important in
evaluating abnormal levels of plasma enzymes. However,
an abnormal isoenzyme pattern may occur despite normal
total activity (see above). The standard unit for enzyme
activity was discussed in Chapter
6
. The normal range
is affected by a variety of factors: age, sex, race, degree
of obesity, pregnancy, alcohol or other drug consumption,
and malnutrition. Drugs can alter enzyme level
in vivo
and
interfere with their measurement
in vitro.
Enzyme activities may also be measured in urine, cere-
brospinal fluid, bone marrow cells or fluid, amniotic cells
or fluid, red blood cells, leukocytes, and tissue cells.
Cytochemical localization is possible in leukocytes and
biopsy specimens (e.g., from liver and muscle). Under
ideal conditions, both the concentration of the enzyme
and its activity would be measured. Radioimmunoassay
(RIA) and its alternative modes such as fluorescence im-
munoassay (FIA), fluorescence polarization immunoassay
(FPIA), and chemiluminescence immunoassay (CLIA)
(discussed
later),
can be
used to
measure enzyme
concentration
as
well
as
other clinically
important
parameters.
M easurement of Enzyme Activity
In the assessment of enzyme levels in the clinical lab-
oratory, the most frequently used procedure consists of
measuring the rate of the enzyme-catalyzed chemical re-
action. The initial rate of an enzymatic reaction is directly
proportional to the amount of enzyme present when the
substrate concentrations are maintained at saturating lev-
els (i.e., zero-order kinetics with respect to substrate con-
centration) and other factors (e.g., pH, temperature, and
cofactors) are maintained at optimal and constant levels
(see Chapter
6
). Under these conditions, the rate of sub-
strate removal or product formation in a given time in-
terval is directly related to enzyme activity. The reaction
rate is determined by measuring product formation (or
substrate removal) between two fixed times or by continu-
ously monitoring the reaction with time. For accuracy, it is
preferable to measure product formation rather than sub-
strate removal because the former involves measurement
of a change in concentration from an initial zero or low
level to higher levels, which is analytically more reliable
than the reverse. Both methods, fixed-time and continuous
monitoring, are kinetic procedures. A schematic diagram
of an enzymatic reaction is shown in Figure 8-2. The true
rate of enzyme activity is obtained only when the reaction
rate is measured in the linear region (a period of constant
reaction rate and of maximum velocity). Inaccurate results
are obtained if the rate is measured during nonlinear or
nonmaximum phases. In fixed-time procedures, in which
a single given interval of time is chosen to measure en-
zyme activity, it is essential to select reaction conditions,
such as concentration of substrate, pH, temperature, and
cofactors, that will provide linearity with maximum slope
during the time period in question. False negative values
for enzyme rates may arise from substrate depletion, in-
hibition of the enzyme by product, increase in the reverse
reaction due to product accumulation, denaturation of the
