Hello everybody,
I´d like to analyse content of cholestrol and bile acids in mouse tissue. I´ve got methods to extract both cholesterol and bile acids from tissue to the point of drying samples under the nitrogen to obtain dry, purified lipid fraction. Which solvents could I use to dissolve cholestrol or bile acids properly and at the same time maintain the function of sensitive enzymatic assays? I´m affraid of alcohols in my assay. EtOH definitely does interfeer with the bile acids enzymatic assay :(
Thank you very much!
J.
Hi Jana. I have a protocol for liver, intestine, and gall bladder bile acid extraction, and lipid extraction from feces which I am happy to email to you (too long to post here).
As for using EtOH, we resuspend/extract bile acids in varied concentrations of EtOH with a final extraction process in 2:1 (v:v) chloroform:MeOH. We have not found that using EtOH or chloroform:MeOH interferes with our assay. We are using Diazyme Laboratories Total Bile Acid Assay, and Calbiochem Cholesterol/Cholesteryl Ester Quantitation Kit.
I will email the protocols to you if you'd like them!
Sebastian and Jessica, thanks to both of you! Jessica, I´ll be glad to read your protocol! My e-mail adress is on my profile.
Hi Jassica Francl ,i am working on gallstone disease i need those protocols please send me to
my mail id : [email protected]
Thank you
I want to estimate cholesterol,bile salts and phospholipids in Gallstones and Bile,
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 dissolving cholesterol, I am using 10% Triton X-100 (for 20mM concentration) you can also use this concentration or varried concentration. In this, you can do, enzyme assay. But I am not sure about bile salt, Most probably it will also soluble in Triton X-100 and do not interfare in enzyme assay.
Hi-I found the best method is to extract the non-polar phase as you described (via Folch or Bligh and Dyer), dry under N2 stream, bring it back up with chloroform plus 1%Trition X-100 , dry with N2 stream once more and then dissolve in water. Works like a charm for me.
Thank you both! I will definitely try this option with triton-X and choloroform and maybe I´ll try to compare it with EtOH and MetOH and post results ;)
Hi Jana,
Did you try the EtOH, MetOH and triton methods and compare them? Can you please share your results?
Thanks,
Chris
Many thanks to all, it's really very informative medium!
Could any one suggest me Buffer composition for cholesterol solubility ? i' m really searching for buffer medium similar to saliva to get dissolved cholesterol, considering the solubility of cholesterol in water is very low 0.095 mg/L (30 °C)? I am afraid of organic solvent for cholesterol dissolution.
Please anyone help me ?
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