Kinetic technique (IFCC METHOD)
a) Basics of kinetic technique:-
Read Principle of Kinetic technique for enzyme testing
b) Applying principle of kinetic technique on the measurement of ALT activity:-
AST (GOT) catalyzes the transfer of amino group (-NH2+) between 2-Oxoglutarate and L-aspartate producing oxaloacetate and L-glutamate. So, the substrates we used in this test are 2-Oxoglutarate and L-aspartate. Then oxaloacetate will react with NADH in the presence of malate dehydrogenase enzyme (MDH) to form NAD+. So, MDH and NADH are required in your reagent.
We said in the principle of the kinetic technique for enzyme testing, that measuring the appearance or increment of products concentration is more accurate. Therefore, we measure AST activity by the rate of appearance or the increment of the concentration of oxaloacetate which is measured by the rate of oxidation of NADH at a wavelength of 340nm (which means decrement of NADH concentration). This means that we measure the rate of increment of oxaloacetate concentration by measuring the rate of decrement of NADH concentration, therefore, After adding the sample to the working solution, we wait for one minute then we read the initial absorbance value. Then, we read the absorbance every one minute till 3 minutes. Thus we, will have 3 readings; A1, A2, A3. Then we subtract each of them with the previous one to get the ΔA (this will be explained more in the calculation section). The rate of decrement of NADH concentration per time is directly proportional to AST activity (i.e. the more the NADH concentration decrease means the more oxaloacetate formation which means the higher AST activity). Therefore, the graph of absorbance versus time is a straight line with negative slope.
- If you have a spectrophotometer, You need:-
- Accurate pipetting devices (Micropipette is the preferred) to prepare 1 ml of the working reagent and to add the sample.
- Cuvettes with 1 cm light path to prepare the working solution and add sample on it, then use it to read the absorbance.
- Timer to adjust it to the time intervals according to the reaction
- Water bath adjusted at 37oC to make the temperature of the reaction is stable at 37oC
- If you have a semi-automated analyzer:-
- Tubes and micropipettes to prepare 1 ml of the working reagent and to add the sample.
- Water bath adjusted at 37oC to make the temperature of WR reaches 37oC before adding the sample.
- Cuvettes, if the semi-automated analyzer has not a suction system.
You don’t need:-
- Cuvettes if the semi-automated analyzer has suction system,
- Timer, because the semi-automated analyzer has its own timer and reads the absorbance according to the time you program on it.
- Water bath or incubator during the reaction, because it has its own incubator which is adjusted at 37oC.
Your reagent should have the following:
- Buffer solution. (in AST test, TRIS buffer is usually used)
- Substrates which are oxoglutarate and L-alanine.
You can buy ready reagents or you can prepare it by yourself and we will introduce the method for preparing this reagent in other topics but for now, we will take 2 examples for ready reagents which are:-
- AST test by the kinetic method by biosystems.
- AST test by the kinetic method by Spinreact.
Here we will talk about the general concept of the procedure.
souNote:- these procedures are for spectrophotometer users, not for semi-automated users
- Prepare the working solution as recommend by the reagent supplier.
- Clean the cuvettes if you use a spectrophotometer or semi-automated analyzer without a suction system or flush the suction system of your semi-automated analyzer if it has a suction system by keeping the tube in a distilled water for 2 minutes.
- Zero the spectrophotometer or photometer with distilled water or air at 340nm. (i.e. the working solution is colorless)
- Incubate the reagent in the water bath at usually 37oC for some time to reach the temperature needed for the enzyme to (NOTE: The needed volume of the reagents, the temperature, and the time of the incubation are recommended by your reagent supplier).
- Then you can add the serum or the plasma. Then start the timer immediately and return the tube to the incubation in the water bath and wait for one minute delay. This delay is to get the first absorbance from which the first-minute reading will be subtracted (see the calculation section for more information) (NOTE: – the volume of the sample and the incubation time is recommended by the reagent supplier).
- After the one-minute delay, read the absorbance after one minute, 2 minutes and 3 minutes (3 readings total). And you should return the solution to the incubation after each reading to keep the temperature of the solution at 37o
- Calculate mean absorbance difference/minute (ΔA/Min.). Then multiply it by the factor that we will calculate in the next section.
- If the sample is hemolyzed, collect another sample and if it is icteric or lipemic, dilute the sample 1 in 10 with distilled water. (how to make dilution and calculate the dilution factor click here)
In the case, if you use a semi-automated analyzer, you will flush the suction system, if it has a suction system, or clean the cuvettes if it doesn’t have a suction system. Then choose the programmed procedure for AST test or ALT test (you program it according to the reagent supplier) (the both programs are allowed because they have the same parameter). In this case, you can prepare the working solution in a cuvette if it doesn’t have a suction system or tube if it has a suction system. Then place the cuvettes or tubes in the thermostable cell holder of the semi-automated analyzer or in the water bath, then read the blank and then add the sample, start reading immediately after adding the sample, where the analyzer starts its own timer wait for 1 minute delay and then takes the 3 readings as scheduled then multiply the absorbance by the factor which we are going to calculate in the calculation section. (if you don’t make the analyzer start reading after adding the sample immediately, you will get false low result) DON’T forget to flush again at the end of the test if it has a suction system. IT IS EASIER THAN SPECTROPHOTOMETER