CREATINE KINASE ISOENZYME AND ISOFORMS

Creatine kinase is a dimeric enzyme that will catalyze the reversible reaction of phosphorylation of creatine by ATP. 

(Source: Tietz Clinical Chemistry, 4th Edition)

The enzyme (CK) in serum is relatively unstable, activity being lost due to oxidation of active site sulfhydryl group of active site. This inactivation can be inhibited by incubating the enzyme with –SH compounds like N-acetylcysteine, dithiothreitol (Cleland reagent), and glutathione.

CK is dimer with 2 subunits (B and M) each with mol. Wt. of 40,000 KDa and it has 3 isoenzymes, CK-1, 2, 3. These are numbered according to their electrophoretic mobility with anodal form receiving the lowest number according to criteria of Commission on Biochemical Nomenclature.

CK exists as cytosolic isoenzyme (CK-1, 2, 3) and mitochondrial isoenzyme (CK-Mt). 

Isoenzymes                 % of CK activity (Source)

q  CK-1 (CK-BB)   -           >90% (brain, intestine).

q  CK-2 (CK-MB) –           1% (Skeletal muscle) 25-30% (Myocardial muscle)

q  CK-3 (CK-MM) –          98% (Skeletal muscle), 77% (myocardium)

q    CK-MM – 3 isoforms

 q  CK-MB – 4 isoforms

CKMM and CKMB isoforms are formed by post-translational modification (cleavage of C-terminal lysine by carboxypeptidase)

Analytical Measurement:

Isoenzymes of CK are measured by using monoclonal anti-CK2 antibodies and rely in immunoinhibition method which will inhibit the M subunit of CK-MB and measures the B subunits. Isoforms measurements are also done by advanced techniques. ELISA methods are also used to measure CK-MB using solid phase anti-MB antibody and using anti-B antibody conjugated with enzyme as detection antibody.

Total CK activity can be measured by enzymatic kinetic method: Also after inhibition of CK-M the CK-B is also assayed by the same process. 

(Source: Tietz Textbook of Clinical chemistry, 4th Edition)

 

 

There is increase in absorbance at 340 nm followed every minutes for 3 minutes and CK-MB activity obtained by multiplying the CK-B activity by 2. N-acetyl-L-cysteine is added as an activator to maintain a supply of reduced sulfhydryl groups necessary for the complete activation of CK. Adenylyl kinase present in serum can produce ATP from ADP producing positive interference so, this enzyme is inhibited by adding diadenosine pentaphosphate or by running sample blank using AK instead of CK.

The reference range at 37°C for total CK is 46-174 U/L for adult men and 96-140 U/L for adult women. The reference range for CK-MB activity is <6% of total CK and CK-MB mass is 0-5 ng/L.

Addition of EDTA will also stabilize the enzyme by chelating with calcium.

CK isoenzymes can also be quantified by electrophoresis: 

CK-MB activity assay have been replaced by CK-MB mass assay. This assay can detect an increased amount of CK-MB about 1 hour earlier than activity based assays. These assay based on sandwich technique where solid phase capture antibody directed to M subunit and detection antibody directed to B subunit. This detects only CK-MB. In addition, calculation of a relative index (CK-MB mass assay/total CK x 100) may be used as an indicator of MI. A relative index >3% is indicative of AMI.