activator, and the measurement of increase in absorbance at 340nm or by fluorescence
polarisation (primary wavelength 340nm, reference wavelength 378 nm):creatine phosphateþADP !creatineþATP
ATPþD-glucose !ADPþD-glucose-6-phosphate
D-glucose-6-phosphateþNADðPÞþ ! D-6-phosphogluconate
þNADðPÞHþHþ- CK-MB activity: This is assessed by the inhibition of the activity of the M monomer by
the addition to the serum sample of an antibody to the M monomer. This inhibits CK-MM
and the M unit of CK-MB. The activity of CK-BB is unaffected but is normally
undetectable in serum hence the remaining activity in serum is due to the B unit of
CK-MB. It is assayed by the above coupled assay procedure and the activity doubled to
give an estimate of the CK-MB activity. An alternative assay uses a double antibody
technique: CK-MB is bound to anti-CK-MB coated on microparticles, the resulting
complex washed to remove non-bound forms of CK and anti-CK-MM conjugated to
alkaline phosphatase added. It binds to the antibody–antigen complex, is washed to
remove unbound materials and assayed using 4-methylumbelliferone phosphate as
substrate, the released 4-methylumbelliferone being measured by its fluorescence and
expressed as a concentration (ng cm^3 ) rather than as activity. - Aspartate aminotransferase activity: This is assessed by a coupled assay with malate
dehydrogenase and the measurement of the decrease in absorbance at 340 nm:
L-aspartateþ2-oxoglutarate (^) !oxaloacetateþL-glutamate
oxaloacetateþNADHþHþ (^) !malateþNADþ
- Lactate dehydrogenase: The measurement of total activity is based on the measurement of
the increase in absorbance at 340nm using lactate as substrate. The measurement of LD-1
is basedonthe use of 2-hydroxybutyrate as substrate sinceonly LD-1andLD-2can use it:
Total LD: lactateþNADþ !pyruvateþNADHþHþ
LD-1:2-hydroxybutyrateþNADþ !2-ketobutyrateþNADHþHþThe clinical importance of obtaining early unambiguous evidence of a myocardial
infarction has encouraged the development of markers other than enzyme activities
and currently two tests are commonly run alongside enzyme activities. These are based
on myoglobin and troponin-I:- Myoglobin: Concentrations in serum, assayed by HPLC or immunoassay, increase
more rapidly than CK-MB after a myocardial infarction. An increase is detectable within
1–2 hours, has 100% sensitivity and reaches a peak within 4–8 hours and returns to
normal within 12–24 hours. However, myoglobin changes are not specific for
myocardial infarction since similar changes also occur in other syndromes such as
muscle damage or crush injury such as that following a road accident. - Troponin-I: This is one of three proteins (the others being troponin-T and troponin-C)
of a complex which regulates the contractility of the myocardial cells. Its activity in
644 Principles of clinical biochemistry