method for quantitative analysis of cholesterol in biomembranes - cholesterol content (Nov/27/2006 )
first, thanks for members reading this topic
i want to ask about how can i detect the quantitation of cholesterol and its
distribution in the tegument of any worms particulary,platyhelminthes. we used filipin
staining method but it is used for visualization only
thanks for your helps
Cholesterol is measured either by chemical or enzymatic direct or indirect spectroscopic methods. Direct methods don't need an extraction step but are prone to interference from bile salts, lipaemia etc. You'll need to work out if the worms have bile salts.
The chemical methods use alcohol in concentrated acid to yield coloured cholesterolenes which are detected spectroscopically at 410nm. You'll need to set up a standard curve.
Enzymatic methods have three reactions; first the chol esters are hydrolysed using chol esterase. Then the free chol is oxidised (chol oxidase). This yields cholesterol ketones and peroxide. Finally, the peroxide is detected by reacting them with aminoantipyrine + phenol to yield quinonamine dye which you detect at 510nm. Although more work, I think this is a better method.
All this can be done in the one test tube. You can either measure in kinetic or end-point mode. In kinetic you measure after say 18 mins. In end-point you let the reaction process finish (straight line reading).
The biggest problem may be the extraction step. You can use ultracentrifugation or an organic extraction (hexane chloroform). There are also HPLC and GLC methods. You need the extraction stage in them, though.
Check out Clinical Chemistry by Tietz for specifics of the methods.
As for the distribution, well, use fluorescent chol probes to bind in situ.
Let us know how you get on and just holler if you need more details.