Title: Clinical Enzymology – Enzymology – Estimation Estimation of Transaminase Material and Method: Refer to the lab manual. Result: Table 1: The following table showed the individual absorbance value of the serum sample. Tube Substrate volume (ml) Incubate at 37 for 3 minute Sterile water volume (ml) Serum sample (ml) Absorbance (A)
1
2
Blank
0.5
0.5
0.5
-
-
0.1
0.1 0.071
0.1 0.076
0
Table 2: The table below recorded the absorbance values of standard Oxaloacetate (OAA) solution. Tube Substrate Volume (ml) Standard OAA volume (ml) Standard OAA concentration (ml) Absorbance (A)
1
2
3
4
5
6
0.50
0.40
0.30
0.20
0.10
0.05
-
0.10
0.20
0.30
0.40
0.45
-
0.20
0.40
0.60
0.80
0.90
0
0.113
0.198
0.281
0.376
0.406
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Calculation: Part 1: To convert the volume of standard OAA solution into concentration The concentration of standard OAA solution in 1ml is 2 mol/ml. To obtain the concentration of OAA solution in 0.1ml, we must use the compare method to get the value which showed as follow: 2 mol/ml: 1ml = X: 0.1ml X = 2 mol/ml x 0.1ml X = 0.2 mol ANS: Therefore, the concentration of standard OAA solution in 0.1ml is 0.2 mol. To calculate the rest of the concentration of OAA solution in 0.20ml, 0.30ml, 0.40ml and 0.45ml, we can use the same method which showed above to obtain their respective values. After undergo a few steps of calculation, the OAA concentration are 0.40 mol, 0.60mol, 0.80 mol and 0.90mol,
respectively.
Part 2: Calculate the activity of AST in serum From the standard curve, the amount of oxaloacetate (OAA) in the patient’s sample is 0.15mol/ml .
The formula for calculating the activity of Aspartate transaminase (AST) in
mol/min/litre of serum (U/L) is:
AST activity in serum =
2
Given: Amount of OAA= 0.15 mol/ml; Time = 30 min; Volume = 0.001L
AST activity in serum =
=
= 50 mol/mol/liter = 50U/L ANS: Therefore, the activity of AST in serum is 50U/L. Discussion: The presence of aspartate transaminase in the serum will act on aspartate when they are incubated together at 37 in a buffer of around neutral pH, to form the respective keto acid which is oxaloacetate. The keto acid will be able to react with 2-4 dinitrophenyl hydrazine (DNPH) in alkaline NaOH solution to form reddish-brown complex of hydrazones. By using the spectrophotometer, the intensity of color is proportional to the amount of oxaloacetate present which in turn is proportional to the amount of serum aspartate transaminase present. Therefore, measurement of optical density of the color solution indicates the concentration of the serum aspartate transaminase levels (Rao, 1992). From the standard curve we have plotted, we can observe that there is a linear relationship between the absorbance and the amount of oxaloacetate (OAA). When the concentration of OAA increase, the absorbance reading will also rises accordingly. This 3
means that the rising of absorbance value is proportional to the OAA concentration which in turn is proportional to the amount of aspartate transaminase (AST) present in the patient’s serum. Therefore, using the spectrophotometer enable us to estimate the patient’s serum AST level. The reference range for AST in a healthy person is 10 – 45U/L (Beckett, 2005). The analytical result obtained from the spectrophotometer for the amount of AST within the patient’s sample revealed that there is a slight increase of AST in the serum which is 50U/L compared
to the reference range. This showed that the AST value is fell out of the
normal range. The escalating of AST concentration in serum may be associated with many of the disease such as myocardial infarction, pancreatitis, hepatitis and so on. Therefore, based on the test the patient has been conducted, we are able to predict the several possibilities of disorder associate with the rising of AST level in the patient.
Estimation of serum enzymes, especially the aspartate transaminase is the most sensitive and useful test for diagnosis of various types of diseases. Many of the possible diseases may be related to serum AST level. Serum AST is a mitochondrial enzyme can be widely found in human tissue. The heart, liver, and skeletal muscle have a high concentration of AST within it whereas only a small amount of AST found in the kidney, pancreas, and erythrocytes (Bishop, 2010). When the body tissue or an organ such as heart is damaged, additional AST will be released into the bloodstream and at the same time, this rising of AST concentration is directly related to the extent of the tissue damage (Aspartate Aminotransferase).
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The escalating of AST amount in the bloodstream may reflect that a patient is having a trauma of skeletal muscle or myocardial infarction. Besides, in a viral hepatitis, high level of AST will also present in the blood. Furthermore, the patient who is an alcoholic may show a rise in AST level as well. This is because heavy consumption of alcohol will develop cirrhosis which means scarring in the liver and this will further become a liver disease (Liver Blood Test). A pregnant woman or a patient who take certain drugs will also cause the increased of AST concentration in that particular person. The other factors that which could initiate the increased of AST level are the pancreatitis and hemolytic of red blood cells. All the factors that we have described above will directly influence the final result of the tested sample which is cause the rise of AST level. In addition, when conducting the phlebotomy, care should be taken because the hemolysis of the RBC will subsequently increase the AST level in the blood sample. Additional Information: For
men,
children ,
AST level are
14 to 20U/L while
for
women ,
AST level are
10 to 36U/L .
For
they have the highest AST level compared to both men and women which are 9
to 80U/L .
Precaution Steps: 1. Before extracting the patient serum from the sealed bottle, we need to wear glove to prevent contracting any infection from the patien t serum. 2. Bear in mind that, the patient’s serum must be stored in the cool box with ic e to maintain the temperature to prevent the enzyme aspartate transaminase contain in the serum been denatured by the room temperature. 5
3. After drawing the serum sample from the sealed bottle, we must quickly put them back to the cool box. 4. The micropipette tips must change frequently after drawing the different types of solution to prevent any contamination to occur. Conclusion: According to the experiment, the activity of AST in the patient’s serum is 50U/L. This value let us know that the patient has a slight high level of AST in his or her serum compared to the reference range given. High level of AST may be due to the various possible disorder and damage of the tissue or the organ. In short, all the expected results have been fulfilled. References: Rao, S.B. (1992). Practical Biochemistry for Medical Students. Academic Publishers. Beckett, G. W. (2005). Clinical Biochemistry (Lecture Notes). (7 Blackwell Publishers
th
ed.). Oxford:
th
Bishop, M.L. (2010). Clinical Chemistry: Techniques, Principles, Correlations. (6 ed.). Lippincott Williams & Wilkins. Aspartate Aminotransferase (AST). Retrieve February 17, 2010 from
http://www.webmd.com/digestive-disorders/aspartate-aminotransferase-ast Nabili, S. T. Liver Blood Test . Retrieve from http://www.medicinenet.com/liver_blood_tests/page2.htm
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