Cholesterol exists in serum both in free and total form. The serum cholesterol test is important due to many reasons. Excessive cholesterol may lead to deaths due to heart attacks etc. Also, excessive cholesterol makes you fat and lazy. This also affects your daily routine since you become more and lazier. Now the estimation of cholesterol in the body with simple tests can be done easily. But when it comes to the estimation of serum cholesterol, we go for more advanced tests such as Cholesterol Oxidase Method. Today, I will you the process of estimation of serum cholesterol by cholesterol oxidase method. I am deeply going to explain the principle, reagents, and procedure of this test. After reading this article, you will have a complete understanding of the test. Let us now begin with our serum cholesterol test by cholesterol Oxidase method.
The principle of Estimation of Serum Cholesterol by Cholesterol Oxidase Method:
In this article, we are going to study two principles for the test. The first principle is for the free cholesterol and the 2nd one is for the total cholesterol. We will first talk about the principle to measure the free cholesterol in the serum.
As we know that free cholesterol is present in the plasma, we need to disrupt these lipoprotein complexes. But this should be done in a particular manner i.e. the free component must be released in a form which is appropriate for enzymatic oxidation. Also, make sure that the associated cholesterol esters are not hydrolyzed in the test. Triton X-100 plays an important role in this case since its role is dual here. It has to t disrupt the lipoprotein complexes along with the holding of free cholesterol. It is later released to serve as a substrate in the cholesterol oxidase method.
The 2nd principle is for the estimation of total serum cholesterol. The reagent used in this test is alkaline ethanol which is suitable for releasing cholesterol which is present in the esters. This also hydrolyzes the esters. After the neutralization of this element, mercury is added to the solution which is a part of an oxidizing agent. The liberated cholesterol will be useful in a way that it gets stored in micellar solution for enzymatic oxidation purposes with the help of Triton X-100. Hydrogen peroxide is then obtained after this step. It is then estimated by using a colorimeter using a Ti4+ component. And this was the complete principle of the total cholesterol in serum estimation.
Now we know about both of the principles that are being used in this test. Let us now tell you about the reagents that we will use in this test.
Reagent for Cholesterol Oxidase Method.
To perform any biological test, we need some reagents that are key in performing these tests. Of course, there are reagents that are prepared by combining different elements. The reagent used in this test is a combined color reagent. This is made by mixing Ti4+ and dilute sulfuric acid.
Serum Cholesterol Test Procedure:
Here are the few easy steps to perform this test.
- The manifolds for the assay of total and free cholesterol in serum can be mounted on the same pump and run either independently, or simultaneously by linking their sample lines to a common sample probe with the use of a stream splitter (Technicon, PT2).
- The range of the free cholesterol channel should be set to be equivalent to a cholesterol concentration of 4.00 mole/liter.
- When operating in a dual-channel mode, cholesterol standards of greater concentration than 6.00 mole/liter, which are required for calibration of the total cholesterol channel, overload the free cholesterol channel.
- To prevent carryover from these high standards affecting the subsequent only standards up to 6.00 mole/liter are placed at the beginning of a test.
- These are then repeated at the end of the run, together with the higher standards in the range for total cholesterol.
So, this was all about serum Cholesterol test by Cholesterol Oxidase Method. If you have any query or feedback about the article then leave us in the comments section below. Keep visiting our website for more biological tests and more.