Thank you..

Try the Wybenda & Pillegi Method.

procedure for estimation of cholesterol used to NIN protocol method

In my opion, animal sterol is called cholesterol, fungal sterol is called ergosterol. We use HPLC to estimate fungal ergosterol from plant litter. We use supersonic and vortex method to separate ergosterol ester from cell wall and saponification to transfer ergosterol ester to ergosterol. Then we use Pentane to extract ergosterol from methanol solvent. And let it to volatilize all night. The next day we dissolve ergosterol again in methanol, and filter then HPLC. This is Gulis method, Stimulation of leaf litter decomposition and associated fungi and invertebrates by moderate eutrophication: implications for stream assessment. Hope to help you.

We used a dispersive solid pahase extraction, followed by GLC-MS analysis for determination of cholesterol in cells.

See: Giera, Ploessl, Bracher, STEROIDS 72, 633-642 (2007)

Franz Bracher, Munich

You can extract the cholesterol in Chloroform:methanol (1:2 v/v). Collect the chloroform phase and dry under nitrogen. Dissolve the residue in propanol followed by enzymatic determination using kits.

first add methanol to sample than keep it in water bath at 60 C then centrifuge and then collect the filtrate, if there is a layer then again add methanol but if not then add benzene. after centrifugation, collect the supernatant and measure absorbance at 450nm

Use rhplc

Thank you to all,

i want to estimate cholesterol in gallstones,

using chloroform am extracting the cholesterol in gallstones and estimating by enzymatic method it is a end point reaction gives pink color , the problem with black stones (pigmented stone) its not giving pink color in reaction instead it showing yellow color its giving a high value than yellow stones (cholesterol stones)...

i want to quantitatively estimate cholesterol levels in black stones (known as pigmented stones)

I used both ELISA and nano drop, i am taking the reading at 505 nm.

help me out

For the enzymatic test, can you leave the enzyme out of one well (or cuvette) and add buffer instead to get a background at 505 nm? This should allow you to subtract the contribution of the pigment (unless the pigment is reacting with the enzyme as well).

To Dr.Christopher Lloyd

sir i am not able get your answer can you please explain

Thank you..

Wybenga and Pileggi's method:-

Cholesterol reacts with hot solution of Ferric perchlorate, ethyl acetate and sulphuric acid ( cholesterol reagent ) and gives a lavender color complex, which is measured at 560 nm. High density lipoprotein (HDL) are obtained in the supernatant after centrifugation. The cholesterol in the HDL fraction is also estimated by this method.

Dear Govardhan Bale:

If you are measuring a product from an enzymatic reaction at 505 nm and the pigments are interfering with that measurement, one way of compensating for that is to do the same reaction with and without the enzyme present - that way in the enzyme-containing reaction you will get the enzymatic product PLUS the interference; in the no-enzyme reaction all the absorbance at 505 nm is from the interference. The contribution of the enzyme product would be calculated from the reading at 505 nm WITH enzyme MINUS the reading at 505 nm WITHOUT the enzyme. The only thing you need to do (to keep concentrations from reactants, interferences, etc the same) is to add the same volume of buffer to the no-enzyme reaction as you used enzyme. This should work unless the interfering substances also react with the enzyme.

Good luck.

Thank you sir

I did this procedure but what my doubt is the calculated value from the reading at 505 nm WITH enzyme MINUS the reading at 505 nm WITHOUT the enzyme is correct or not ?

Because what i am thinking is the reaction color and sample color mix and gives change in the end reaction color that i will read and take the value

Our working protocol in the LAB, perhaps this also works with the stones, good luck

Cholesterol in Bile.

Aim

The method for free cholesterol employs cholesterol-oxidase to generate H202 and peroxidase to catalyze the reaction of H2O2 with Parahydroxyfenylacetic acid to yield a stable fluorescent product measurable at Emission 415nm / Exication 325 nm

Cholate enhanced the sensitity of the signal ( Allain C, Clin.Chem 1974, 20: 470-475)

Reagents:

• Phosphate-buffer, 0.2 M, pH=7.4

10.9 gr K2HPO4.3H2O

1.8 gr KH2PO4

in 250 ml bidest

• Na-Cholate, 20mM, pH=7.4 ( 430 mg Na-Chloate in 50 ml Phosphate-buffer pH=7.4)

• Triton-X100, 0.5%

• Para-hydroxyfenylacetic-acid, Sigma H4377

• Cholesterol-Oxidase Roche 126934

• Peroxidase Roche 108073

• Ethanol ( EtOH) , abs

• Cholesterol-stock solutions 40ug/ml (0.103mM) Cholesterol in EtOH

Step-by-Step Procedure

Making of the Assay Mix, prepare freshly:

• 12 mg Para-hydroxyphenylacetic acid, Sigma H4377

• 14 ml Phosphate-buffer, 0.2 M, pH=7.4

• 1 ml Na-Cholate, 20 mM, pH=7.4

• 3 ml Demi

• 1 ml Triton-X100 ,0.5%

• vortex

add:

• 120 ul Cholesterol-oxidase,

• 2.4 ul Peroxidase,

• 10 ul Cholesterol-esterase if nessesary

• mix carefully ( do not vortex).

The Assay:

Sample pre-preparation:

Make a calibration curve :Pipette 0; 25; 50; 75 and 100 ul stock solution (40ug Cholesterol/ml EtOH) into a glass tube, make the final volume 100 ul whith EtOH abs

Use 15 ul Bile, extract the lipids Bligh&Dyer,

Resolve the lipids in 450 ul Chloroform , split and use 300 ul (=10 ul bile) for CHOL-determination and evaporate.

• Dissolve the Samples or Standard in 100 ul EtoH absolute before use

• Add 600 ul Assay Mix

• Mix Gently

• Incubate in de dark for 20 min at room temperature

• Transfer 200-250 ul to a white or black 96-wells Elisa plate

• Measure the intensity at 415/325 nm

Calculations

Make a calibration curve :Pipette 0; 25; 50; 75 and 100 ul stock solution (40ug cholesterol/ml EtOH) into a glass tube, make the final volume 100 ul whith EtOH abs

Use a linear calibration curve to calculate the cholesterol in the samples

.

References

Gamble W, Vaughan M, Kruth HS, Avigan J. Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells. J Lipid Res. 1978 Nov;19(8):1068-70

Allain C, Clin.Chem 1974, 20: 470-475