Fatty acid oxidation assay - conversion Oleate to CO2

[Ugo51]

Hi guys,

I'm doing some in vitro fatty acids oxidation assay following the protocol by Sweeney 2005 (Diabetologia 48: 132–139).

Basically I trap the radiolabelled CO2 with filter paper which was previously soaked in phenetylamine:methanol and then I count the activity of each piece fof filter paper.

 

To get also the CO2 that is in solutution in the medium (bicarbonate) I acidify the medium with H2SO4 to pH1. It kills the cells of course, but at that point I no longer care

 

My point is: one of my collegues insists that she puts the H2SO4 on the filter paper together wiht the phenetylamine and than she incubates the flask (sealed) with this paper on the lid.

Is there any reason why she should acidify the filter paper instead of the medium?

 

I tried, it didn't work, nor I see any explanation why the H2SO4 onthe filter should be of any use.

 

Are these two different methods, or only one is right?

(and I'm pretty sure to know which one...)

 

 

thanks a lot

 

 

[John Cuthber]

In the presence of strong acid (H2SO4 or others) the phenylethylamine will not absorb CO2.

 

So counting the CO2 absorbed on it will be pointless (you will get little or none).

[Ugo51]

Thanks.

Just to have a better understanding of the process, how does the phenethylamine bind the CO2?

onto the C adjacent to the N in form of Carboxylic acid?

[John Cuthber]

Probably a few reactions but the easy one is this

CO2 + 2 R-NH2 + H2O --> (RHN3)2 CO3
I.e. the reaction to give the carbonate.

 

 

The bicarbonate is also a possibility if there's a lot of CO2 and I wouldn't rule out the formation of carbamates

http://en.wikipedia.org/wiki/Carbamate

[Ugo51]

so, basically, in presence of the strong acid the CO2 doens't get trapped because all the phenethylamine forms a salt with the acid itself, am I right?

 

I guess I will have a hard time explaining this to my workmate given she doesn't know much of chemistry, but I can give it a try

 

thanks for your help!