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Help with lipid peroxidation assays


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11 replies to this topic

#1 cardosopedro

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Posted 24 August 2011 - 10:01 AM

Hey!

Anyone in the forum with experience with lipid peroxidation? Would you say that lipid peroxidation occurs as an early or as a late event following ROS production?


Thank you.
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#2 zienpiggie

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Posted 24 August 2011 - 01:21 PM

Hi Cardosopedro,

Lipid oxidation is quite a complex event, but the peroxyl radical commonly generated during lipid oxidation is one of the slowest reacting radical of all type of radicals, but at the same time it has the longest half lives.

It also depends on the fatty acid composition in question, thus what kind of lipid material you are trying to oxidize. Samples containing higher level of unsaturated fatty acids will oxidize faster as opposed to that containing more saturated fatty acids.

In addition, the type of lipid oxidation end point measure will also make a difference. As you most probably know, lipid oxidation begins with lipid oxidation initiation, followed by propagation and termination. Different assays will measure lipid oxidation at different stage of lipid oxidation. So whether it is early or late will also depends on how you are measuring them.

I am not sure if this helps.. but I hope it does.

#3 cardosopedro

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Posted 25 August 2011 - 07:49 AM

Sure it helped! Thank you.

I am using two methods: the TBARS assay (reaction with thiobarbituric acid) and free MDA assay (reaction with 1methyl-2-phenylindole).

I see production of ROS very early after drug treatment (I am using a cell death-inducing drug). However, I don't seem to find lipid peroxidation even trying very different time points of analysis... Posted Image I tried from 2h to 24h of treatment. Maybe... it simply does not happen??


Thanks again.

#4 zienpiggie

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Posted 25 August 2011 - 09:05 AM

I tried TBARS as well before on my cell extract and it wasn't working so well. It is likely because TBARS is not a very sensitive method. I have seen however some people being successful in using the assay. If you are working with cell extract, may be you might want to concentrate your samples or try something else more sensitive? You might also want to use positive control that you know for sure will oxidize your samples. AAPH for example is a free radical generator that is reported to induce lipid oxidation.

For me I eventually resorted into using a probe called DPPP that will be incorporated in cell membrane and react with lipid hydroperoxides. The assay eventually worked but looking for instruments that measure the specific fluorescence for the probe wasn't easy. Another way I was thinking of trying but never got around to do was to try gas chromatography for the secondary oxidation product such as hexanal.

#5 cardosopedro

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Posted 26 August 2011 - 07:15 AM

Thank you for joining the discussion. I actually did a positive control. I used H2O2 and it worked. So, the method is working... The problem is that I can't see differences in my samples of interest (both with TBARS and free MDA assay), although I see production of ROS...

#6 zienpiggie

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Posted 26 August 2011 - 04:47 PM

Just wondering, what is the magnitude of ROS generation in your drug treatment as opposed to H2O2? And compared to H2O2, is it produced slow and steadily or in an instant?

#7 cardosopedro

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Posted 27 August 2011 - 03:14 AM

I never measured ROS after H2O2 in these cells...

Mmmm... but regarding stimulation of ROS production by my drug, I would say that it happens quickly. I already see a very significant difference when comparing with the control after 30 mins of treatment.

The earliest time point I tested for lipid peroxidation was 2 hours after treatment. Now I am wondering if it can occurs very early (I mean, before 2 hours) and I am missing it because of that.

If this is true that would mean that lipids "loose" its peroxidation state (because I don't see peroxidation at 2 hours)?


Thanks for the nice discussion.

#8 zienpiggie

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Posted 27 August 2011 - 09:30 PM

HI Cardosopedro,

I am wondering of how did you measure the ROS?

I think measuring the lipid oxidation at 30 min might be a good idea but then to me it does not seem like it makes sense that you would 'lose' lipid peroxidation. MDA are secondary product of lipid oxidation so the lipid oxidation likely has gone all the way passing the propagation stage. Once it is there, unless if there is additional antioxidants, it seems that observing continuous generation of MDA is more likely.

Also, I wonder of what is the longest incubation time that you have tried?

#9 cardosopedro

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Posted 29 August 2011 - 12:59 AM

I measured ROS by flow cytometry using two probes: dihydrorhodamine 123 and dihydroethidium.

For the lipid peroxidation assays, the longest treatment with the drug was 24 hours. I will try this week one of the assays after 30 mins.

#10 zienpiggie

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Posted 29 August 2011 - 09:50 PM

Hi Cardosopedro,

just an idea, do you think it would be a good idea to compare the ROS generation pattern of your drug as opposed to H2O2? Another question is whether you know if your drug compound fluoresces at nearby wavelength that your probes are fluorescing at? A reason to look at the ROS pattern of your drug vs H2O2 is that cells have their own defense system that can neutralize the radicals, so if they got neutralized quicker than H2O2, I thought may be it prevent lipid oxidation from occurring. My two cents.

#11 cardosopedro

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Posted 31 August 2011 - 03:02 AM

zienpiggie,

I just tried the TBARS assay after 30mins of treatment and... no luck. Posted Image

How common is for the cells to start producing ROS without lipid peroxidation as a consequence? Is it plausible that the increase in ROS can simply function as signaling not having other consequences like the peroxidation?

#12 cardosopedro

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Posted 31 August 2011 - 03:05 AM

Thanks again for your opinion.

Yes, I will perform a catalase activity assay to see if I have differences in scavenging.

Regarding the other question... my drug fluoresces but at a very different wavelenght (both excitation and emission) than the probe does.

Edited by cardosopedro, 31 August 2011 - 03:05 AM.





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