Hi Masood,

That's a very good question.

If you have sufficient resources, measurements taken from all three types of samples will give you more meaningful data than just looking at one sample type. I would suggest to look even closer at different cellular compartments of your tissue samples, for e.g. cytosol, mitochondrial matrix, and peroxisomes. As different redox states have been measured at different cellular compartments at one single time point. There are kits out there to help you isolate these compartments for your analysis.

However, if you have limited resources, looking at tissue itself will be the most direct measurement and allows you to draw the most relevant conclusion from your findings. The more distant the measurements (interstitial fluid, serum, etc), the more confounded your measurements will be due to "contaminations" by oxidative stresses generated from other sites or organs.

Hope that helps.

Hi Masood,

That's a very good question.

If you have sufficient resources, measurements taken from all three types of samples will give you more meaningful data than just looking at one sample type. I would suggest to look even closer at different cellular compartments of your tissue samples, for e.g. cytosol, mitochondrial matrix, and peroxisomes. As different redox states have been measured at different cellular compartments at one single time point. There are kits out there to help you isolate these compartments for your analysis.

However, if you have limited resources, looking at tissue itself will be the most direct measurement and allows you to draw the most relevant conclusion from your findings. The more distant the measurements (interstitial fluid, serum, etc), the more confounded your measurements will be due to "contaminations" by oxidative stresses generated from other sites or organs.

Hope that helps.

I agree with Kai Lun Chang. If possible try to do with all the samples. But to standardize the protocol to serum is a very difficult task.

Even serum based analysis of antioxidants is one good approach because all expressed proteins and enzymes are released in it, but has certain limitations as few high molecular weight proteins which otherwise masks the presence of small test molecules. Besides, specific tissues are better candidates being directly affected of oxidative stress and expresses ample antioxidants in response to it. Doing it with multiple tissues always keep your statistics at its high and fetch you good publication.

You may like to homogenize the organ, make a supernatant, dissolve the supernatant via liophilization, and then check any component of the antioxidant system.

You make sure to standardized the procedure which will you follow and take a look Follow serum based analysis for antioxidant, because it is approachable and effective way to your findings.

Sorry to express dissent, but i dont agree with Kai at all.

Isolation of subcellular components alters the redox situation they were in, when they were inside the cell. Isolation is indeed possible even without kits. what you need are some silica sols (ficoll percoll) and a good high speed centrifuge with a swing out rotor. However, once you isolate organelles, their redox context changes. The interior ofthe cell is highly reducing while the environment outside is oxidizing.So, to put it simply, you may isolate and estimate the machinery that generates/maintains antioxidant status, the redox status of the antioxidants themselves will be altered. Please read about GSH for instance. It has to be derivatized immediately to allow detection of GSH/GSSG ratios. Please also remember that all antioxidants always exist as redox couples, unless they are structurally sacrificed in the course of action. Therefore, the risk is that evry estimation you make will give you some reading and may make you happy. While the situation of redox balance at the time you opened the cell up may have been extremely different.

As far as serum is concerned, you need to work fast and if possible , use nitrogen to keep oxygen out before you make the estimations. keeping your vials stoppered and working fast is the least expensive method indeed for sera.

I would strongly advise you to consult the landmark publication in this area by Halliwell and Gutteridge before you embark on practicals. There are several excellent reviews in the area that you may search and consult. I am sorry if my comment appears to be critical and falsely knowledgable. It is not intended to be so.

However, having worked in the area for some time, I have found to my consternation, that the area of antioxidant research is at the same time very easy to explore and very difficult as well. Easy as all the assays are simple and largely inexpensive, so everyone can perform them. Extremely difficult, since if one performs them without taking note of the oxidizing atmosphere we work in, there are a million pitfalls and the result we obtain are most likely to be wrong. Truly, "bliss of ignorance"seems to work overtime here.

The best way of assessing the redox status of the cell is live cell imaging with fluorescent molecular probes using a very good (and extremely expensive) fluorescence of LSC microscope. The microscope as well as the probes are way out of reach for not-so-well funded labs, to put it very gently. Fluorimetric estimations using whole cells and a multimode plate reader are a close second. of course you have the option of flow cytometry also.

Dear Samir,

Thank you for your comment. I appreciate your candid opinion.

I completely agree with you that one has to be careful when analyzing subcellular components, especially in this case where redox states or reactive species will interact with its environment.

Nevertheless, analyses of subcellular components could still generate useful data if the right measurements are taken. For e.g., SOD and NOS are enzymes with different levels of subcellular localizations, and their varied expressions under oxidative stress had been studied for many diseases.

As Masood did not specify the type of measurement he plans to take, I gave a very general recommendation. I understand there are certain caveats in my previous post, and I thank you for pointing them out.

Your question is intresting.

If you want to check effects of stress on antioxidant defense system, best organs is brain, because brain is directly affected organ by stress.

I already published a reseach paper which helpful you to find effect of stress on anti-oxidant parameter. Please find attachemnt for same

You can also use any orgasn like liver, pancreas etc. or fluid like serum, CSF to measere the anti-oxdiant parameters like glutathoin, SOD, catalase, lipid peroxidation.

I would like to add my opinion to the type of tissue to be used for determination of anti-oxidants. The first affected organ by the oxidative stress is the lung tissue which in turn sends the signal to brain which later on gets affected.

I therefore suggest to do the comparative studies of antioxidant status on all three tissues, viz., lung, blood and brain.

Hello Samir,

Your question is related to Toxicity. First of all what kind of tissue did have test it. For example Lung tissue means. You will take small quantity of lung tissue for evaluation of Oxidative stress. Thiobarbituric acid reactive substances (TBARS) is most methods for evaluating oxidative stress. Just try this method . Defintely you will get good results about Oxidative stress measurement. Follow this reference ; Nichans and Samuelson, 1986)

I wish him for success.

As per your protocol you take organs, that to be exocrine and endocrine organs, even you can do it with serum

Dear Masood,

Your question does not mention the specific organ you are interested in, so the following might not work for you. Breath analysis offers a way around issues such as research ethics since it avoid invasive procedures such as tissue or blood sampling. If you can think of an experiment that would stress (more or less) specifically the organ you are interested in, you could analyse exhaled breath for markers of oxidative stress, typically products of lipid-peroxidation. On the up-side such a protocol gets around pitfalls of in-vitro studies already pointed out by previous correspondents since you are observing in-vivo systems and doing so using a non-invasive method. On the down-side, such a set-up still contains an element of a black box so your experimental design has to anticipate as many of the potential sources of artefacts to ensure your test achieves the necessary sensitivity and specificity to tell you something meaningful. Another problem with sampling breath with the aim to detect and quantify biomarkers such as hexanal (and other markers such as isoprene) is the fact that these compounds are susceptible to (aut)oxidation, which means the old Tedlar bag to collect breath samples won't cut it since one needs stainless steel flasks that are electro-polished on the inside so as not to offer any active site that could catalyse or trigger (aut)oxidation of captured biomarkers during sample storage prior to analysis. Prof Michael Phillips has published extensively on the subject of analysing breath for volatile biomarkers, so it might prove helpful to contact him and pick his brains on this. [email protected]

Dear Kai,

Point taken. I agree. This is what is best about Researchgate. The area developes as we talk about it and all of us learn. I always have. Thanks, people.