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chapters
Enzymes III: Clinical Applications
reaction must be the rate-limiting step. The indicator
reaction should be capable of instantaneous removal of
the product formed in the primary reaction to prevent or
minimize reversal of the primary reaction. Maintenance
of a large excess of the second enzyme (malate dehydro-
genase) in the reaction mixture fulfills this requirement.
The indicator reaction must function at the concentration
of the substrate (oxaloacetate) of the indicator enzyme, so
that the rate of the indicator reaction is directly propor-
tional to substrate concentration. Under these conditions,
the rate of reaction catalyzed by the indicator enzyme is
directly proportional to the rate of product formation in
the primary reaction. Thus, during the initial phase of
the reaction, the indicator reaction must wait for a pe-
riod of time known as the
lag phase
for the primary
reaction to generate adequate quantities of oxaloacetate
to permit the maximum rate of conversion of NADH
to NADU
In nicotinamide adenine dinucleotide-linked assays,
endogenous metabolites and enzymes in the serum may
cause oxidation or reduction of the coenzyme, thereby
introducing errors. For example, in the AST assay, the
NADH of the indicator reaction may be oxidized by the
presence of pyruvate and of LDH in the serum:
Pyruvate + NADH + H+ U L-lactate + NAD+
This reaction falsely raises the measured activity of AST.
A way to prevent this is to include a preincubation step
in the assay procedure. This step consists of a time pe-
riod during which all components of the reaction mixture
except one of the substrates of the primary reaction (in
this case, a-ketoglutarate) are allowed to incubate. This
period permits all undesirable reactions to reach comple-
tion before the primary reaction is initiated by addition of
«-ketoglutarate. Because of the need for rapid and ac-
curate measurement, automated procedures have been
introduced.
8.2 Serum Markers in the Diagnosis
of Tissue Damage
Myocardium
Coronary artery occlusion causes heart tissue damage
due to ischemia and can lead to
myocardial infarction
(MI). The immediate and common cause of artery ob-
struction is formation of a thrombus. Anti thrombolytic
therapy (Chapter 36), with streptokinase or recombinant
tissue plasminogen activator, protects the myocardium
from permanent damage by restoring of blood flow. An
early diagnosis of acute MI (AMI) is crucial for proper
management. A patient’s history, presence of chest pain,
and electrocardiograms are problematic in the diagnosis
of AMI. Therefore, measurements of circulatory proteins
(enzymes and nonenzyme proteins) released from the
necrotic myocardial tissue are useful in the diagnosis of
AMI.
Characteristics of an ideal myocardial injury marker are
cardiac specificity, rapid appearance in the serum, sub-
stantial elevation for a clinically useful period of time and
ease and rapidity of the analytical essay. At present, no
serum marker fulfills all of these criteria. Some cardiac
markers that appear in plasma are myoglobin, LDH, CK,
and troponins. Measurements are made at the appropriate
time intervals based upon the appearance of the marker
in the plasma (Table 8-1). Frequently utilized markers
are CKMB and cardiac troponin I (cTn I). Troponins
consist of three different proteins I, C, and T, and are
expressed in both cardiac and skeletal muscle. The tri-
partite troponin complex regulates the calcium-dependent
interaction of myosin with actin (Chapter 21). Troponins
are encoded by different genes. Cardiac I and T isoforms
have unique structural differences from their skeletal mus-
cle counterparts. However, cTn T like CKMB undergoes
ontogenic recapitulation and is reexpressed in regener-
ating skeletal muscle and in patients with chronic renal
failure. CKMB is also present in the skeletal muscle, al-
beit in small concentration. Compared to the myocardium
(about 360- 400 g), skeletal muscle consists of a much
larger mass (about 40% of total body mass). In rhabdomy-
olysis (disintegration or breakdown of muscle) CKMB
and myoglobin appear in plasma in significant quantities.
Myoglobin, although an early marker (Table 8-1), lacks
specificity, and CKMB is elevated in many circumstances
other than cardiac injury, e.g., rhabdomyolysis, chronic
renal disease, and degenerative diseases of skeletal muscle.
CKMB has a subform in the plasma due to the fact the M
subunit undergoes cleavage of a lysine residue from the
carboxy terminus by the plasma enzyme carboxypepti-
dase N. Tissue and the plasma subforms of CKMB have
been designated as CKMB2 and CKMB1, respectively.
The ratio of the serum levels, CKMB2/CKMB1
can also
yield information useful in the early diagnosis of MI. Mea-
surement of LDH isoenzymes (LDH1 and LDH2) also
lacks specificity; they appear significantly later after the
myocardial injury and show ontogenic recapitulation. Car-
diac troponin I, a highly specific marker for AMI, does
not suffer from these disadvantages and appears in the
plasma as early as CKMB and persists as long as LDH
isozymes. For these reasons, serial serum cTn I mea-
surements may be superior in the detection of MI even
in patients with chronic renal disease, rhabdomyolysis,
or diseases of skeletal muscle regeneration (e.g., muscle
dystrophies).
