2.4 - Creatine Kinase

Question 1

Creatine kinase reaction

Answer 1

creatine phosphate <–> creatine + ATP

• catalysed by creatine kinase
• ADP + H+ –> ATP

Question 2

Where is creatine kinase found?

Answer 2

• CK present in all cells but present in particularly high concentrations in metabolically active tissues e.g. skeletal muscle, brain and heart
• following damage or death of such cells CK is released into circulation
• three dimeric isoenzymes of CK are known in humans - there are two different subunits M and B
• MM = skeletal muscle
• MB = cardiac muscle (myocardium, where 15% MB, 85% MM)
• BB = brain muscle
• the three isoenzymes can be separated by electrophoresis on cellulose acetate strips - MM moves furthest towards negative electrode

Question 3

What is a myocardial infarct?

Answer 3

• the death of heart muscle cells, due to a lack of oxygen
• lack of oxygen due to blockage of cardiac arteries - process termed atherosclerosis

Question 4

Why do cells need oxygen?

Answer 4

• semi-permeable membrane separates inside from outside of cell, actively excluding some things e.g. Na+
• this requires a protein pump in the membrane (ATPases) which use energy in the form of ATP
• ATP is generated via glycolysis, the Krebs cycle and eventually oxidative phosphorylation
• the end point of the process requires atmospheric oxygen, hence if there is less oxygen supplied to a cell there is less ATP, pumps do not function, ion balance is lost and cells die

Question 5

When and why is creatine kinase found in the blood?

Answer 5

• cell contents are released when they are dying –> proteins that should be held inside against concentration gradients appear in the serum
• therefore the levels of many proteins including CK (and others e.g. lactate dehydrogenase) in serum can be used as indirect indicators of cell death

Question 6

How might you determine creatine kinase activity?

Answer 6

• CK activity in the serum can be detected by a coupled assay leading to the generation of detectable products
• remember NADH (and NADPH) have absorption spectra distinct from NAD+ (and NADP+)
• creatine phosphate + ADP –(CK)–> creatine + ATP
• ATP + D-glucose –(hexokinase)–> ADP + G6P
• G6P + NADP+ –(G6PD)–> 6-PG + NADPH + H+

Question 7

Why might the three isoenzymes be separated by electrophoresis?

Answer 7

• CK is a protein made from two subunits or monomers - dimer
• the two monomers are coded for by two different genes
• these generate two different monomer isoforms - ‘B’ and ‘M’
• the two monomers have approximately the same molecular weight but differ in their isoelectric point (pl) = can be separated by charge

Question 8

How might one establish a diagnosis of myocardial damage?

Answer 8

• if both genes are expressed in a cell, three final dimers are possible - BB, MM, MB
• the brain only expresses the B gene = only makes B monomer so only BB form generated
• conversely the MM form is only one made in skeletal muscle cells - useful for diagnosing extent of skeletal muscle damage in MD
• the only tissue where both genes are expressed is cardiac muscle cells = make all three dimers, including the hybrid MB form
• thus, death of cardiac muscle fibres can be determined if the MB isoform of CK can be detected in serum

Question 9

Does an increase in serum CK activity relate to the size of the myocardial damage?

Answer 9

• levels of CK-MB in serum are directly proportional to the amount of cell death in the heart
• this is because each myocyte can be considered to be approximately of equal volume (equal likelihood of dying independently of their size)
• so, as each cell dies it releases a ‘quantum’ of CK into the extracellular fluid and thence into serum

Question 10

What is the time course of serum CK after a myocardial infarction?

Answer 10

• CK - increases steeply and peaks 24 hours after, then decreases
• SGOT - increases and peaks 48 hours after, then decreases (slightly shallower decrease)
• LDH - increases shallowly, peaks around 5 days after, then decreases shallowly

Question 11

What other markers can be used for diagnosis of myocardial damage?

Answer 11

• SGOT - serum glutamate oxaloacetate transaminase
• LDH - lactate dehydrogenase
• cardiac troponin - cardiac troponin I and troponin T only present in the heart - appearance in the serum is a specific marker for cardiac infarction (typically appearing in the serum 48h after infarction and persisting for approximately 5 days)