CLINICAL CHEMISTRY 2 – LABORATORY HANDOUT AY 2011-2012
EXPERIMENT NO. 7 - ACID PHOSPHATASE
1. Discuss the principle of the test.
ACP catalyzes the hydrolysis of Paranitrophenylphosphate, which is colorless, at an acidic pH, to paranitrophenol, which is colored yellow, at 450 to 470 nanometers.
2. Why do we utilize the mentioned wavelength for ACP?
To be able to get the maximum reading of ACP in the sample.
3. What is the ideal specimen for the determination?
Citrated blood is ideal, but for the determination performed in the lab, it is serum.
4. Name sources of errors for ACP test
Hemolyzed serum falsely increases values
Turbid and icteric serum need serum blanking for accuracy
Some reagents are photosensitive, exposure to light would decrease values
Prolonged or shortened incubation time would increase and decrease values respectively
Alkaline pH would decrease values
Altered temperatures could either increase or decrease values
EXPERIMENT NO. 8- ALKALINE PHOSPHATASE
1. Discuss the principle of the test.
ALP catalyzes the hydrolysis of Paranitrophenylphosphate, which is colorless, to paranitrophenol, which is colored yellow, at 405 nanometers.
2. Name differences between ALP and ACP
| category | ALP | ACP |
| pH | Basic or alkaline | acidic |
| Best specimen | Heparinized plasma (Calbreath) | Serum citrated plasma (Calbreath) |
| Tissue source | Same as ACP except for prostate, more on bone clin. significance | Prostate, platelets, bone, liver, spleen, kidneys, erythrocytes |
| Diagnostic significance | Hepatobiliary and bone disorders | Prostatic carcinoma |
3. What is the reason for diluting serum if the absorbance is higher than 0.25?
For more precise and accurate measurement of the concentration of the unknown.
4. Why do we have to adjust the spectrophotometer to zero when we read unknown solutions?
To read out errors caused by the spectrophotometer and the reagent.
5. What is the best sample for this determination?
Unhemolyzed, clear, non-icteric, non-lipemic serum
EXPERIMENT NO. 9 – LACTATE DEGYDROGENASE DETERMINATION
1. Discuss the principle of the test.
LDH in the serum catalyzes the oxidation reduction of lactate to pyruvate, which is measured spectrophotometrically.
2. Give the reasons why serum LD determination cannot be refrigerated.
Because LD isoenzymes are thermolabile and are unstable at refrigerated temperatures. They would not be able to react accurately.
3. Why should there be timed intervals in the addition of 0.1N HCL?
So that the acid could react properly with the LDH in the sample.
4. Why should the incubation period be done exactly in 5 minutes?
Because incomplete reaction would occur if it is less than 5 minutes and more products would be formed when prolonged more than 5 minutes. This is at specified temperature and conditions.
5. Name sources of errors in this determination.
Hemolyzed serum increases result 100-150 X
Turbid, lipemic and icteric serum needs serum blanking for accuracy
Refrigerated blood samples lowers values
Prolonged or shortened incubation time at specified conditions could increase or decrease values respectively
Altered temperatures could either increase nor decrease values
EXPERIMENT NO. 10 – ALANINE AMINOTRANSFERASE DETERMINATION
1. Discuss the principle of the method.
ALT in the serum catalyzes the transfer of an amino acid group to a keto acid group to form pyruvate and L-glutamate. This is measured spectrophotometrically at 505-535 nanometers.
2. Aside from sodium hydroxide, what reagent could also be used to alkalinize the solution?
Potassium hydroxide
3. Why should the distilled water used in dissolving your NaOH pellets be CO2 free?
Because carbon dioxide can affect the transfer of the amino group to the keto acid group due to its carbon content. The carbon atom may act as an acceptor molecule in the reaction. This would falsely decrease your values.
4. Why do we use a semi-log graphing paper in plotting your calibration curve?
Because the it is simpler to use since the graphing paper will find the logarithms of the values beforehand.
EXPERIMENT NO. 11 – ASPARTATE AMINOTRANSFERASE DETERMINATION
1. State the principle of the test.
AST in the serum catalyzes the transfer of an amino acid group to a keto acid group to form oxaloacetate and L-glutamate. This is measured spectrophotometrically at 505-535 nanometers.
2. Name sources of error in this method.
Hemolyzed serum will increase values 10-15 X
Turbid, lipemic and icteric serum needs serum blanking for accuracy
Altered temperatures could either increase nor decrease values
Prolonged or shortened incubation time at specified conditions could increase or decrease values respectively
3. What is the purpose of allowing the reagents to come to room temperature.
The purpose is to allow the reagents to inactivate the reagents so that they could react properly.
4. Differentiate AST from ALT. Cite specific differences.
| CRITERIA | AST | ALT |
| Substrate | L-aspartate & alpha-ketoglutarate | L-alanine & alpha-ketoglutarate |
| Old name | SGOT | SGPT |
| One of the end products | oxaloacetate | Pyruvate |
| Amount in inside serum | 10-15 times | 5-8 times |
| Major clinical significance | heart | Liver |
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5. What is the component of the SGOT substrate?
L -aspartate and alpha-ketoglutarate
