1. The document discusses measuring creatine kinase (CK) levels in the blood of a healthy person and a cardiac patient to detect muscle damage. 2. CK levels were higher in the cardiac patient (214.7 U/L) compared to the healthy person (97.62 U/L), indicating possible myocardial infarction in the patient. 3. CK is released into the blood from damaged muscle tissue and can be used as a biomarker for detecting cardiac muscle damage like heart attacks.

1. Department of Biochemistry and Molecular Biology
University of Dhaka
MS Practical
Mesurement of Cretinine Kinase from Blood of a Cardiac patient
Submitted by
Md. Atai rabby
MS

2. Priciple
The development of sensitive spectrophotometers able to measure a wide range of wavelengths has
allowed natural substrates and measurements in the ultraviolet region of the spectrum to be used in
clinical tests. Assays using natural substrates and measurements in the ultraviolet region are the
basis of most of the commonly used methods to determine enzyme activities in clinical
biochemistry. Typically, they continuously measure the absorbances of NAD+ or NADP+ at 340
nm. Figure shows the reactions used to monitor the activities of creatine kinase (CK) in clinical
laboratories by measuring changes in the absorbance of NAD+ or NADP+. Clinical investigations
using this enzyme can be used to detect muscle damage..

3. Table : Absorbance of Sample from Healthy and Cardiac Patient at 340nm
Time (min) Absorbace at 340 nm
Healthy person Cardiac Patient
0 0.077 0.117
1 0.090 0.153
2 0.105 0.183
3 0.121 0.217
Calculation
For Healthy Person, Absorbance change per miniute
∆A1 = 0.090 – 0.077 = 0.013
∆A2 = 0.105 – 0.090 = 0.015
∆A3 = 0.121 - 0.105 = 0.016
Avarage ∆A/min = (∆A1+∆A2+∆A3)/3 = 0.015
Cretine Kinase Activity = ∆A × Calibration Factor = 0.015 × 6508 = 97.62 U/L
For Cardiac Patient, Absorbance change per miniute
∆A1 = 0.153 – 0.117 = 0.036
∆A2 = 0.183 – 0.153 = 0.030
∆A3 = 0.217 - 0.183 = 0.034
Avarage ∆A/min = (∆A1+∆A2+∆A3)/3 = 0.033
Cretine Kinase Activity = ∆A × Calibration Factor = 0.033 × 6508 = 214.7 U/L

4. Result
Cretine Kinase Activity in healthy person = 97.62 U/L
Cretine Kinase Activity Cardiac Patient = 214.7 U/L
Comment
1. The CK activity of healthy person lies in normal range [ 24-190 U/L at 370
C ]
2. The CK activity of the patient is High than reference value indicating possible Miocardial
Infraction But further diagnosis in necessary for conformation.
Discussion
A biomarker is a biological molecule whose concentration in the blood changes in response to a
specific disease. Biomarkers are normally considered in relation to a specific type of tissue damage.
Biomarkers that indicate muscle damage,in particular damage to cardiac muscle, which can lead to
heart disease are usually referred to as cardiac biomarkers.
A number of different types of molecules have been exploited as biomarkers. These include:
 enzymes and their associated coenzymes or cofactors
 structural tissue proteins
 intermediates of metabolic pathways
 messenger molecules
Creatine kinase transfers a phosphate from creatine phosphate to adenosine diphosphate (ADP) to
form adenosine triphosphate (ATP) . When muscle tissue contracts it consumes ATP; however, in
some conditions muscles can potentially run out of ATP. Should this happen, the muscles would
then stop working. However, muscles have stores of creatine phosphate that in the short term can be

5. used to phosphorylate the ADP to ATP. The released creatine is subsequently broken down to
creatinine, whichis used to test for renal function.
The level of CK activity in the blood is due to leakage from muscle tissues. This means that its
activity in the blood will be affected by muscle mass and muscle composition. This is the reason
that the reference value for CK activities in the blood of men is higher than that of women, who on
average have less muscle mass. Slightly different types of muscle fibres are found in the muscles of
different ethnic groups, which explains, for example, why CK levels are higher in Afro-Caribbeans
than Caucasians.
Creatine kinase is found in all the muscles of the body. Its activity in the blood begins to increase
about 4–6 hours following an AMI and peaks at 24 hours. However, there is much more skeletal
muscle than cardiac muscle in the body. Consider someone involved in a road traffic accident who
has a crushed chest and thus a damaged heart. There is also skeletal muscle damage, so the increase
in CK from the damaged muscles may mask the increase resulting from the damaged heart.
However, CK activity can be due to one of three isoenzymes.
CK1 or CKMM, found mostly in skeletal muscle
CK2 or CKMB, found predominately in cardiac muscle
CK3 or CKBB, found in smooth muscle
Assaying the individual isoenzymes can help in distinguishing between the CK activities from
cardiac muscle and skeletal muscle damage. Thus, measuring CKMB activity is a more sensitive
method of detecting an AMI, especially if there has been damage to skeletal muscle.Creatine kinase
MB activity starts to increase slightly earlier than the overall CK levels and peaks 21 hours after a
MI. To better distinguish between cardiac and skeletal muscle damage the amount of CKMB can be
expressed as the ratio CKMB/total CK. The theory is that if the ratio increases it is more likely to be
cardiac than skeletal muscle damage.
The usefulness of CKMB in detecting AMIs has led to the development of immunoassays for its
measurement. However, there is a drawback to the use of CKMB in detecting AMI.

6. Although cardiac muscle contains more of the MB than other isoenzymes of CK it also contains
significant amounts of CKMM. Similarly, although skeletal muscle also contains mostly CKMM, it
does have significant amounts of CKMB. Crucially, this means that an increase in CKMB is not
absolutely specific for cardiac damage, although for many years it was the best available test.
In addition to the isoenzymes of CK, there are also isoforms of CKMM and CKMB. Isoforms of a
protein differ from one another due to post-translational modifications. Post-translational
modifications include chemical modifications, such as glycosylation and phosphorylation, and, as is
the case here, the removal of certain amino acid residues. Isoforms of CKMM and CKMB are
formed by a deaminase in the bloodstream that removes the carboxy terminal amino acid residue
from the M subunit of CK molecules. This means that there are potentially two isoforms of CKMB,
called MB1 and MB2, and three isoforms of CKMM, called MM1, MM2, and MM3. There was
initial interest in using the ratio of MB1 to MB2 as a very early test for AMI although this has been
replaced by assays for cardiac troponin. However, measuring isoforms of CKMM is potentially a
useful way of determining whether there has been recent skeletal muscle damage. The common
form of CKMM is MM3, so an increased amount of MM1 would suggest that there has been recent
skeletal muscle damage. This test is not yet used in routine clinical practice.
Heart disease occurs due to blockage of the blood vessels supplying oxygen to the heart muscle. In
any tissue where the need for oxygen from the blood exceeds the rate at which it can be supplied, a
deficiency of oxygen or ischaemia occurs. However, ischaemia is reversible provided it is not
unduly prolonged. When the ischaemia is prolonged, irreversible cell damage and cell death can
occur; this is known as infarction, and is followed by cellular breakdown and necrosis. Thus,
infarction is irreversible.
In the heart, ischaemia of cardiac muscle will occur if the artery supplying blood to an area of
cardiac muscle becomes partially or totally blocked. The reason that a partial blockage can also
cause ischaemia is that some areas of cardiac muscle are on the borders of the area supplied with
blood by the arteries, areas called watersheds. In a normal state they receive just enough blood to
survive, but should one of the arteries become even partially blocked then the already barely
adequate supply to this muscle slips below the minimum level; thus the muscle becomes
inadequately supplied with oxygen and slides into a state of ischaemia. The ischaemia will be

7. exacerbated if the need for oxygen increases at the same time, for example if heavy exercise is
being undertaken.
A number of terms are used to describe patients with a real or suspected heart attack, which is a
myocardial infarction (MI). Myocardial infarction and acute myocardial infarction (AMI) are
associated with cardiac pain and the death of cardiac tissue (myocardial cell necrosis). In some
patients, such as diabetics, an infarction can occur without pain. It is then referred to as a silent MI.
Angina is often confused with MI but the term simply means heart pain. The difference between
angina and MI is that damage to cardiac muscle does not occur in the former. Angina occurs in two
principal forms: stable and unstable anginas. In stable angina, the cardiac pain occurs predictably
and gradually and can be controlled by actions as simple as physically resting or by using
appropriate drugs. Cardiac pain or breathlessness that is associated with exercise and relieved by
rest is also stable angina. In contrast, in unstable angina the cardiac pain comes on unpredictably
and is not relieved, or only partially, by rest or by drugs.