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Mesurement of cretinine kinase from blood of a cardiac patient

The development of sensitive spectrophotometer 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..

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Mesurement of cretinine kinase from blood of a cardiac patient

  1. 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. 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. 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. 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. 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. 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. 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.

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