Oxidative Stress Biomarkers - Science topic

Thank you Azadeh Eshraghi for your answer.

In my opinion, there are no simple methods to evaluate antioxidizing capacities of potential antioxidants. General tests like DPPH, ABTS... only (roughly) measure the ability of the antioxidants to scavenge free radicals. An antioxidant is much more than that; it can act as a scavenger of free radicals, repair of the oxidized species (reduction by one electron transfer) or by cascade (cumulative) effect . Furthermore, the antioxidant capacity can also depend on the agent responsible for the oxidative stress conditions (selective antioxidants).I recommend the evaluation of the mutual behavior of both antioxidant and the target to be protected in every specific oxidizing environment.For details see e.g.:

Spectrophotometric methods to optimize the amount of prote have been investigated in the following article

Hello J.D. Franco-Navarro

Your question can’t be answered as @Fahad Shafiq has correctly pointed out at the end of the comments because there is almost complete lack of information about the methods. The study seems to have been done with rather small plants, and in such a way that there would be a strong interaction between nutrition and water supply, making interpretation very difficult. Let us assume you grew the plants in soil, applied a liquid fertilizer and allowing some plants in each nutrient treatment to dry by stopping watering, while the control was given ample water. That is a standard molecular biology/biochemistry approach to such studies.

Which was the treatment supplying the most nutrients - A I suspect, with C supplying least. So plants in A would grow better than in C, and have more, larger leaves. When you started the drought treatment plants in A would dry the soil much faster than those in C – simply a matter of greater surface area in A. So plants in A would become severely drought stressed before those in C, which would appear to grow and be much more `drought resistant’. They would also have very different contents of metabolites. The lesson from this – if my supposition is true - is that it IS ESSENTIAL TO COMPARE PLANTS AT THE SAME RELATIVE WATER CONTENT. If this is not done then the study is invalid. I suggest my paper explains the problem and ways of doing such studies correctly – it focuses on GM plants but the problem is absolutely the same for all studies involving water supply.

Lawlor DW. Genetic engineering to improve plant performance under drought: physiological evaluation of achievements, limitations, and possibilities. J Exp Bot. 2013 Jan;64(1):83-108. doi: 10.1093/jxb/ers326. Epub 2012 Nov 16.

By ignoring the complexities involved with water supply in solid matrices and the effects of different rates of water loss, studies of the metabolism, genetic modifications etc are invalid. Hope this rather strong critic does not apply to your study. Please provide the necessary information about methods.

Best wishes

David

Possibly changed parameters are GSH, and a ratio of GSH:GSSG, SOD, MDA, plasma antioxidant vitamins, GPx and catalase.

Thanks Dr. Luay & Dr. Sarah

I get bad experience about using Spectrophotometric analyses in MDA, GSH and antioxidant vitamins

So I didn't agree in past with colleagues who suggested the SPA

I also have a same problem with plasma and serum samples. Some of the samples react to produce color while others failed to develop color. I could not understand why it happens.

Mediators of Inflammation Volume 2015, Article ID 524291, 6 pages http://dx.doi.org/10.1155/2015/524291

Research Article

Urinary Malondialdehyde Is Associated with Visceral Abdominal Obesity in Middle-Aged MenUrinary

MDA was measured with high performance liquid chromatography (HPLC). Urinary MDA levels were expressed as μmol/g creatinine, averaged, and used for analysis.

Hey Harald,

We are trying to both. 8-OH-G appears to be fine, but we really wanted to get the 8OHdG and F2ISO in the same run. May have to do a couple runs in the end. Thanks for the suggestion.

CHeers,

Daniel

Hi David, I am working at the University fo Glasgow on Japanese quail too, with a main focus on oxidative stress. Please send me an email if you want more info, but for methods to measure oxidative stress from red blood cells, you can have a look at most of my papers on RG. I measured protein carbonyl, DNA damage, SOD, GPx, glutathione (total and oxidized).

All the best,

Antoine

Interesting question Safia..

The cellular changes induced by either high temperature  or low temperature  include responses those lead to the excess accumulation of toxic compounds, especially reactive oxygen species (ROS). The end result of ROS accumulation is oxidative stress]. In response to high temperature  the reaction catalyzed by ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) can lead to the production of H2O2 as a consequence of increases in its oxygenase reactions . On the other hand, LT conditions can create an imbalance between light absorption and light use by inhibiting the activity of the Calvin–Benson cycle. Enhanced photosynthetic electron flux to O2 and over-reduction of the respiratory electron transport chain (ETC) can also result in ROS accumulation during chilling which causes oxidative stress. Plants have evolved a variety of responses to extreme temperatures those minimize damages and ensure the maintenance of cellular homeostasis. A considerable amount of works have explored that there is a direct link between ROS scavenging and plant stress tolerance under temperature extremes . Thus, the improvement of temperature stress tolerance is often related to enhanced activities of enzymes involved in antioxidant systems of plants. Plants exposed to extreme temperatures use several non-enzymatic and enzymatic antioxidants to cope with the harmful effects of oxidative stress; higher activities of antioxidant defense enzymes are correlated with higher stress tolerance. Different plant studies have revealed that enhancing antioxidant defense confers stress tolerance to either  high temperature  or low temperature  stress .

Like annual crops, perennial crops are also sensitive to extreme temperature. Fruits and nut trees are important crop plants which often face extreme temperature stress induced damages. Every fruit tree species has a range of optimum temperatures  above or below which the growth and yield markedly reduced. The mean temperatures range for optimum growth of most tropical fruits are about 24-30°C . For instance, mango (Mengifera indica) tree can tolerate high temperature up to 48°C only for a certain period of time , on the contrary it has only partial tolerance to low temperature  In another study, Schaffer et al. observed that monoembryonic mango cultivars tend to be more low temperature  tolerant than polyembryonic cultivars . However, several studies have shown that low temperature promote reproductive morphogenesis in mango. Dinesh and Reddy studied the responses of fruit trees to temperature and observed differential responses to temperature in different fruit species. They concluded that lychee and longan require a warm sub-tropical to tropical climate that is cool but also frost-free or with only very slight winter frosts not below -4°C, and with high summer heat, rainfall, and humidity. In longan, stressful temperatures of <15°C at the young fruit stage reduce fruit growth potential and final size as reported by Young et al.. Stressful  low temperature  also induces excessive fruit drop and severe fruit cracking ....

Dear Mir Alam,

Oxidative stress can be measured using any C. elegans strain. Our group uses DCF-DA as a fluorescent probe to measure ROS accumulation, not specifically H2O2. I just tried the previous method for N2, CF1038 and GR1307, but I guess it would also work for Alzheimer’s-related-strains, such as CL2006, CL4176, etc.

Any of the ROS/RNS kits should work as well as DCF. Another good indicator of OS would be oxidized and reduced glutathione levels (GSSG/GSH)

Thank you Mukesh,

I will check glutothione peroxidase can be done on NMR,

Katie

Gabriela, see in the attached file a paper that we published. 

Best regards

José

I think the important aspect is not comparing the OD between control (Pyrogallol) and samples. It is obvious that the OD of samples will be greater than that of the control. The important thing is looking at the mean difference per minute. If still the mean difference of samples are still higher, then try to look at your buffer (Tris 0.05M EDTA 0.001M pH 8.5). You can try 20mM pyrogallol.

Best regards

UPR/ER stress is activated through an orchestrated interplay of signaling molecules from all three arms of the UPR pathway i.e. IRE1, ATF6, and PERK. This is why, it is critical to analyze signaling players in the context of the mentioned fact. Novus' ER Stress / UPR Antibody Pack contains sample size vials of the top markers namely: GRP78/HSPA5, pSer724 IRE1 alpha, total IRE1 alpha, XBP1, ATF6, and CHOP/GADD153. For scientific technical answers to over 15 similar questions, I would suggest reviewing our UPR and ER Stress FAQs at https://www.novusbio.com/support/upr-and-er-stress-faqs

Thank You sir (Mr. Vrajesh Patel). I will al ways be grateful to you.

Thank you for your helps.~!

Luminol can be measured by chemiluminesence and gives both extracellular and intracellular ROS. Lucigenin is another probe. 

Similarly you can try to use DHE and DCF but these are sepcific for either superoxide or hydrogen peroxide, whilst Luminol will measure all ROS.

The way in which you will have to quantify it mught be challenging. Perhaps express as per number of cells (e.g. relative light units per million cells or absorbance per number of cells.

The regular dose of MitoTracker Deep Red is 25 nM (50 nM already gives near saturation fluorescence), with 30-min incubation at 37C. 

Your mitochondria (live) may have been overloaded. In fact all mitotrackers (green, red) work around this dose range, if your microscope lasers are efficient enough. 

**

As for staining fixed mitochondria, it is not recommended based on chemical knowledge of the tracker structures. All commercial mitochondria trackers carry a positive charge nested in a lipophilic benzimidzole structure, meaning the targeting of these dyes is dependent on mitochondrial membrane potential. In fixed cells, there is no mitochondrial membrane potential to start with, and the staining results are confounded. 

**

Lastly, some dyes have phototoxicity (they could either induce it directly or sensitize other molecules to become photoreactive and damaging). The deep red core is different from that of the red and green trackers. Therefore, there's no sure way to say that one dye's behavior will predict that of another. 

I recommend you reduce the deep red tracker dose and do the staining in live cells (followed by fixing if needed, and check if the the localization is retained). 

Good luck

Thank you Dear Makarova

All the afore mentioned antioxidants have been shown to decrease systemic blood pressure in animal,but their efficacy in humans has not been proven. Mitochondrial oxidative stress seems to play a major role in the pathogenesis of both systemic and pulmonary hypertension. Recent studies have also shown that ROS produced by the mitochondria induce the activation of NADPH oxidases, leading to ROS induced ROS formation and oxidative stress.Targeting antioxidant directly to the mitochondria has been challenging and can produce harmful effects. Studies are ongoing to target mitochondrial oxidative stress.

Measurement of oxidative stress involves direct as well as indirect methods. Many exciting new techniques are now available for measuring ROS directly like electron paramagnetic resonance, histochemical staining methods, Mass Spectroscopy, cytochemical methods, ROS tracer dyes, etc.  Non-invasive techniques using real time imaging of redox changes have been used in animal studies. Kits for estimating enzymes like SOD, GPx, etc are available. Estimating GGT may be a quick and easy to measure enzyme for oxidative stress.

I would do same enzyme activity assay for those samples to see the changes. 

I'll try to look some specific reference for your suggestion.

Thanks a lot

Stefano

thank you very much for your kind help

Regards, Stefano

If you know the number of cells and extract out entire SOD (along with other enzymes) from cells, obviously you can measure activity more precisely. Accordingly, you can divide over all activity (in terms of nanomoles or micromoles of superoxide anion radicals dismutated) with number of whatever cells you have taken.  

However, if you want to measure in vivo, theoretically Yes, but practically it may be impossible for following reasons: (1) Most importantly, how would you take care of supplying same quantity of superoxide anion radicals to both infected and control cells? Can superoxide anion radicals penetrate across the membranes?; (2) Would infection promote generation of superoxide anion radicals (generally any stress would enhance the generation of superoxide anion radicals)?

The serum could be quenching some of the ROS and thus decreasing your DCF signal. You could try staining cells with DCF either for the last hour of your treatment or afterwards (if you can remove the treatment after the 24h). Or are you trying to measure ROS throughout the 24h treatment?

Amplex Red is another reagent that measures H2O2 production, but it is best for measuring ROS in the media (eg. if H2O2 diffuses out of the cell, or if extracellular superoxide dismutates into H2O2). As Jeff and David mentioned, DHE reacts mainly with superoxide. If you want to identify the precise species of ROS, certain methods are better than others (eg. DHE by HPLC versus flow cytometry, roGFP for ratiometric analysis using microscopy...). 

Dear Debaprasad Koner, 

I would recall the same issues suggested by Pierre, the only difference that in our lab a 20% TCA and therefore TCA/TBA ratios varied from the mentioned  (~1/1) without problem. The issue with dissolving TBA deserves special attention, especially because it also changes the fluorescence levels depending on time and preparation procedure.  In addition to that: 

1- You could consider to add an antioxidant as BHT in your homogenates to prevent further MDA production during sample preparation.

2- You could add an emulsification solution as SDS to the assay to improve reaction rates. (If your samples contain relevant fat amounts I would also dilute them and measure by fluorescence instead).

3- You could consider a final extraction with a solvent as N-butanol to measure absorbance/ fluorescence only in the fraction really reactive of your assay.

Please, find attached a protocol which we have been using for a while with high reproductibility.

All the Best

Abel

Dear Ali, I send the link to a publication that could serve as a basis for resolving questions.

Sincerely

Guillermo Schinella

And other one: http://www.nature.com/nm/journal/v19/n6/full/nm.3224.html

As stated by other ROS are shortlived and this is this short life (high reactivity) that explains their deleterious nature. Even H2O2 is short lived if catalase is present.

I find a little bit surprising to imagine that a "somehow measurable pool of ROS" in the homogenate would represent the oxidative stress status in the living matter rather than revealing possible interactions between the homogenate components and the oxygen of surrounding atmosphere. And this without taking into account the questionable nature of "ROS probes".

I acknowledge this is not "optimistic"! Then what I would suggest is to consider possible damages or molecules derived from ROS attack that would integrate the ROS attack in vivo. Possible parameters are:

- GSH/GSSG ratio

- Aconitase/fumarase ratio

- Oxyblot of proteins.

- modification of DNA bases, breaks...

- Oxidized lipids.

Then the history of the homogenate should be taken into account, for example in our experience the Aconitase/fumarase ratio is affected by freezing/thawing.

I hope it helps somehow.

FB

Please see this paper

More and more labs are using GSH:GSSG ratios as an indicator of oxidative stress.  Measuring reduced glutathione, GSH, is fairly easy as at contains a free thiol group.  We use Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid (DNTB)) that reacts with GSH resulting in a product that can be measured at 412 nm.

 GSSG is more difficult as it is present at a much lower concentration than GSH and it needs to be reduced in order to be measured. We add a thiol scavenger to our samples to bind with GSH to make a pyridinium salt.  This prevents GSH from binding to DNTB and from oxidizing to GSSG.

 We split each sample into two tubes; one for GSH and one for GSSG.  The GSH sample can be measured with Ellman’s reagent in the traditional way. The GSSG sample needs to have the thiol scavenger added (preferably at the time of collection).  DNTB is then added followed by glutathione reductase to convert GSSG to GSH.  While the scavenger is still present, the reaction rate for DNTB is much faster.  The sample can then be measured at 412 nm.

This link has additional information on the method as well as protocols for cuvette and microplate assays.  http://www.oxfordbiomed.com/gshgssg-assay-1

More and more labs are using GSH:GSSG ratios as an indicator of oxidative stress.  Measuring reduced glutathione, GSH, is fairly easy as at contains a free thiol group.  We use Ellman’s reagent (5,5'-dithiobis-2-nitrobenzoic acid (DNTB)) that reacts with GSH resulting in a product that can be measured at 412 nm.

 GSSG is more difficult as it is present at a much lower concentration than GSH and it needs to be reduced in order to be measured. We add a thiol scavenger to our samples to bind with GSH to make a pyridinium salt.  This prevents GSH from binding to DNTB and from oxidizing to GSSG.

 We split each sample into two tubes; one for GSH and one for GSSG.  The GSH sample can be measured with Ellman’s reagent in the traditional way. The GSSG sample needs to have the thiol scavenger added (preferably at the time of collection).  DNTB is then added followed by glutathione reductase to convert GSSG to GSH.  While the scavenger is still present, the reaction rate for DNTB is much faster.  The sample can then be measured at 412 nm.

 This link has additional information on the method as well as protocols for cuvette and microplate assays.  http://www.oxfordbiomed.com/gshgssg-assay-1

In my opinion there are other methods to measure Oxidative stress. Lipid markers are F2-Isoprostanes, MDA, Lipid hydroperoxides, 4-HNE. 

8-isoprostanes, 4HNE, MDA, protein carbonyls, nitrotyrosine, dityrosine. 

Dear JK,

The attached file is a recent published review that covers the answer to your question.

Context.—Radiation is a key arm in the multidisciplinary treatment of patients with head and neck squamous cell carcinoma. During the past 2 decades, significant

changes in the way radiation therapy is planned and delivered have improved efficacy and decreased toxicity. Refined approaches in the application of radiation and chemoradiation have led to organ-sparing treatment regimens for laryngeal and pharyngeal cancers and have improved local and regional control rates in the postoperative, adjuvant setting. The molecular and genetic determinants of tumor cell response to radiation have been studied, and several potential biomarkers are

emerging that could further improve application and efficacy of radiation treatment in head and neck squamous cell carcinoma.

Objective.—To discuss the current understanding of potential biomarkers related to radiation response in head and neck squamous cell carcinoma.

Data Sources.—Existing published literature. 

Conclusions.—Several potential biomarkers are actively being studied as predictors and targets to improve the use and efficacy of radiation therapy to treat head and neck squamous cell carcinoma. Several promising candidates have been defined, and new markers are on the horizon.

This review has highlighted several biomarkers that are actively being studied as predictors and targets to improve the use and efficacy of radiation therapy to treat HNSCC. Several promising candidates have been defined, and new markers are surely on the horizon. Biomarkers that lead to significant improvements in patient outcome will require rigorous validation and prospective testing.

Hoping this will be helpful,

Rafik

Dear Arbace,

gGLYCys activity determination in red cells is difficult becouse it is very low. So if you must measure it by spectrophotometry it is a problem. In fact Hb which adsorbs at 340 make impossible this determination in this kind of biological matrix becouse you have to use Hb separation procedures and/or enzyme purification.

On my opinion you should follow this procedure but it requires HPLC separation of the products and fluorescent detection. However even if it is time consuming it can be applied in the presence of Hb requiring only the absence of relevant gGT activity (which is exactly the case of red cells by the way). We use this method.

C.C. Yan, R.J. Huxtable, J. Chromatogr, B. Biomed, J. Chromatogr. B Biomed.

Appl. 672 (1995) 217–224.

Briefly, a solution containing 10 mM ATP,

150 mM KCl, 2 mM K3EDTA, 20 mM MgCl2, 10 mM glutamic acid,

2.5 mM cysteine, 0.5 mM DTT in 1 M Tris/Cl, pH 8.1, was let to

incubate at 37 C for 15 min. Thereafter, 10 ll-aliquots of sample

were added to 150 ll of the incubation solution; after 5 min at

37 C, the sample was deproteinized by 1:1 (v/v) addition of

12% (w/v) TCA containing 1 mM K3EDTA. 0.1 ml of the obtained

supernatant was then reacted with 2.5 mM (final concentration)

mBrB in the presence of 2 M Tris (25 ll). After 10 min of incubation

in the dark at room temperature, samples were acidified by

addition of 5 ll of 37% (v/v) HCl and analyzed by HPLC for the

thiol content as above described.

Dear Chibuike C Udenigwe:

Many thanks for your answer!

Could you be so kind to give me an example of conditions that can lead to further oxidation?

Best regards,

I agree with Dr. Vidal and Dr. Mukherjee. It's better to induce endogenous ROS, rather than using an exogenous ROS to augment them, because species like H2O2 have broad and nonspecific effects if applied globally. Even GSH will be reacted upon. It's hard to say what's being mimicked. 

Your question involves three different enzymes, so the discussions and answers might not be easy to follow. I would suggest you to ask a different question for each enzyme, next time.

Below are some references for catalase and superoxide dismutase, with detailed protocols. I could not find a PDF for the oldest references, but the papers with more recent modifications of the protocols might be more useful anyway.

As for para-oxydase, were you referring to the PON1 enzyme? I attached a few papers on that as well.

I also link to other threads in researchgate with similar questions.

All the best with your work!

Peroxynitrite, superoxide could be very relevant. Myeloperoxidase could be very good source of HOCl in vitro.

Under normal conditions the OGG1 protein half life is much longer that what is reported in the paper cited by Christian. As far as I can judje from our own RNAi experiments, OGG1 half-life in HeLa cells must be >10 hours. We checked the protein expression by Westen blot (normalised by actin). One needs to wait 40-48 hours to obtain a significant knockdown.

To your second question: there is no reason why the lifetime should differ between the 326 Ser and Cys variants, at least under normal conditions. However, under stress this might be different. Some believe that the Cys variant is more prone to oxidation, but I do not know any data showing that that would promote protein degradation.

Dear Dr. Scheckhuber,

Thank you very much for your suggestions.

Hi,

Comet assay detects single strand breaks, please see my profile to get all the information that you need.

Hi Levi,

I'm not sure what the basis of your search is of course, but if it is from a molecular point of view these reviews might be interesting to you: 

This review by Claudia Cremers and Ursula Jacob is very informative: http://www.jbc.org/content/early/2013/07/16/jbc.R113.462929

If you're new to redox signalling and biochemistry, this perspective might be very useful to you as well (it might help to interpret many of the data out there): http://www.ncbi.nlm.nih.gov/pubmed/21459321

Also, we have recently written a review on disulphide-dependent signalling that includes some examples of (disulphide-mediated) redox signalling events that might be of interest: http://www.ncbi.nlm.nih.gov/pubmed/25109988 

Finally, this is quite a long one but there might be some leads in there for you: http://www.ncbi.nlm.nih.gov/pubmed/24634836

Best,

Marrit

Not new but superoxide dismutase activity can be utilized as an antioxidant parameter.

What do you want to do? Why do you need new oxidant and antioxidant parameters? 

Cheers, Christian

I think it is the singlet oxygen or super-oxide molecules that are produced due to single electron reduction and consequently leaking of electron from complex III of mitochondrial electron transport chain. A little amount can be produced from complex I also. Production of superoxide is a direct consequence of transferring electrons from one complex to another complex in mitochondrial electron transport chain.

HE is not really cytosolic, because of its positive charge, marking it easy to penetrate membrane and prone to bind DNA. Prolonged incubation of HE results eventually in a heightened background of fluorescence that is not contributed by specific reaction with superoxide. Therefore overincubation should be avoided. HE preferentially localizes to nucleus in the end points. 

MitoSOX has been recommended by Invitrogen to be used at doses between 1-5 uM. At 5uM, for over 30 min, the probe becomes visiaully toxic to some cells, and triggers off more ROS production, artificially. 

In imaging, MitoSOX also contributes to some (nuclear) DNA staining if incubated for 30 min or longer. 

In theory, an ROS probe can work like a scavenger towards some ROS, esp. when its probe concentration is relatively low to yield a detection fluorescence (e.g. below 1 uM).

MitoSOX is recommended to be used at about 2-.2.5 uM, 15 min to 20 min. 

Mitochondria have their own DNA, so specificity of MitoSOX has been in debate. 

See: 

Transition metal ions in their free state bring unwanted biological oxidation's generating  reactive oxygen species and hence oxidative stress. The Coordination of such free metals in body with certain bio-ligands can modify the redox potential of these free metal ions which can block their redox reactions leading to the production of ROS. This methodology can be indispensable in prevention of metal based oxidative stress by blocking the redundant bio-redox reactions.

a ref can be

Bubbles could be not good for tissues and isolated organs.

As option, oxygenate your solution in other reservoir and recirculate solution via membrane filter to your small bath.

I notice that your sample images contain fixed cells. Under this condition, there's no way for fixed cells to produce ROS (notably superoxide, then H2O2) in logically predictable ways.

It's more likely the fluorescence increase is due to auto-oxidation of the probe, in the presence of laser photon. In other words, photo-activated auto-oxidation. 

This makes the probe not to be called robust enough for imaging. 

****

Our lab has just published a JACS paper on superoxide detection. 

You may consult your supervisor, whether it's desirable to write and request the probe from our lab. We distributed a requested aliquot to a US lab, just one week after the paper was published. 

However, the decisions of whether to grant the request rest on my boss' discretion.... :)

As light halothane anaesthesia lowers sympathetic tone, you will measure lower levels of plasma catecholamines.

GST as an enzyme has many detoxifying functions not related to oxidative stress, so it can change completely independently.

Dear Musa

Important biomarkers of oxidative stress in the brain are lipid peroxidation, protein carbonylation and nitrosylation, DNA and RNA oxidation and the ratio between reduced and oxidized glutathione (GSH/GSSG).

Lipid peroxidation markers widely used include 4-hydroxy-2,3-nonenal (4-HNE), acrolein, F2‑isoprostanes and thiobarbituric acid-reactive substances (TBARs; such as malondialdehyde (MDA)). These are really useful since the brain is enriched in polyunsaturated fatty acids (PUFAs) that are highly susceptible to peroxidation.

Protein oxidation markers include protein carbonyls and 3-nitrotyrosine.

8‑hydroxyguanosine (8OHG) and 8‑hydroxy-2-deoxyguanosine (8‑OHdG) are markers of RNA and DNA oxidation.

These products are used as markers of oxidative stress in human brain and you can find several papers showing their presence in human postmortem brain tissue.

Since endogenous antioxidants and antioxidant enzymes are frequently increased as a mechanism of protection against the oxidative insult they are also used as markers for the occurrence of oxidative stress. 

We have used NBT from Sigma, Himedia, Merck, SD-Fine chemicals etc. NBT from all these sources worked equally well.

As far as NBT assay is concerned for measuring oxidative stress is concerned, I have reservations. NBT can be reduced by number of biological events, almost all dehydrogenases can reduce Tetrazolium. I am attaching a good publication, wherein researchers reported that NBT can even generate superoxide anion radicals (many of us use it for measuring superoxide anion radicals).

It is not clear, what exactly you want to measure (what aspect of oxidative stress)? Are you interested in measuring total antioxidant capacity or lipid peroxidation or levels of enzymatic & non-enzymatic antioxidants?

Dear Leonardo,

thanks so much for Your kind reply. I'll try. I'll contact You if further help is required.

Happy Easter!

Stefano Falone

Dear Joshua

It depends on the question you need to answer. For example for the patients that I follow, protein based biomarkers (or sometimes functional assays looking for the functionality of these proteins) are fundamental for the phenotypic diagnosis of a disease, and genotypic markers are important for genetic counseling.

Moreover, some genes present mutations affecting different domains of the related protein causing astonishing different phenotypes (e.g.: STAT1 loss of function mutations, that can be recessive or dominant with susceptibility to different pathogens, and gain of function mutations with other different phenotypes).

So, for some diseases the genotypic-phenotypic correlations are not so direct and both assays are important to understand the biological phenomena.

     

 Hi

I test this assay the results very good

Hi Shaloam,

MitoSciences offers several kits to assess mitochondrial abundance and activity.  This kit: http://www.abcam.com/Complex-I-Human-Protein-Quantity-Dipstick-Assay-Kit-ab109722.html will allow you to quantify mitochondrial complex I, which can be an appropriate proxy for total mitochondrial protein content.  You can browse around their website for other options too.

Alternatively, you can perform a Western blot for TOM20 or other mitochondrial markers as a proxy for mitochondrial protein abundance.

If you have a fluorescence microscope and analysis software that can quantify fluorescence signals, you can also stain your cells with MitoTracker Green and quantify the signal as a measurement of total mitochondria.

I have used each of these in the past with good success.  All of these approaches are pretty straight forward and routinely accepted in the literature as indicators of mitochondrial abundance. 

I would suggest to reach out to Prof. Dietmar Fuchs (dietmar.fuchs@i-med.ac.at) from Innsbruck Medical University, Austria. He is one of the world's experts on neopterin and has pioneered the development of several assays to measure neopterin. I am sure he will be very helpful. Good luck!

Thank you very much for you answers

I agree with Pascale. Quantification of isoprostanes intended to reflect their production for a long period, representing production chronically. Therefore, it is convenient measurement in 24-hour urine stored at 4 ° C / if possible) and supplemented with BHT to prevent oxidation of the molecules of interest. Regarding the measurement method, I also agree with Pascale is preferred GC / MS, with all its drawbacks for HOMEWORK and derivatization of samples, but is that Elisa available, in my opinion and experience, do not offer nor good practicability and good accuracy of results

i'm now preparing a minireview about this sbject and i'll publish it soon, anyway it's a good idea need to be more and deeply investigated

8-isoprostane is supposed to be a good marker of phospfolipid peroxidation.

Hi Matthew

do you have a protocol for generating oxidized BSA that you could share with me?

cheers

dave

There is no clear relationship between GSH levels  and the realm of 'antioxidant' enzymes. GSH biosynthesis is under the control of the NRF2 system, as are some of the peroxidases, not all. For sure, the 8 GPxs, the 6 peroxiredoxins and the 3 SODs are not regulated the same way. Read the relevant literature before starting meaningless measurements (small collection attached).

Best regards

Leopold Flohé

Yes there is a correlation. Use ETC inhibitors and check the modulation in ROS using mitosox red. Also simultaneously monitor the changes in antioxidant levels. You will definitely get your answer.

Regards

Unless you have a hypothesis that applies to all 8 groups, a 2-way is appropriate.  That is, if you have a hypothesis that group 1 will have a positive effect, but groups 2-7 do not, then you can test with one-tail.  However, you may be missing out on unexpected differences if you don't use two-tail.

TBARS are thiobarbituric acid( TBA) reactive substances. They are different types of aldehydes. due to the presence of reactive oxygen species in the cells lipid peroxidation occurs. Intermediates of lipid peroxidation will ultimately converted to different types of aldehydes ex formaldehydes, acetaldehydes etc. Malondialdehyde is the end product. So malondialdehyde is the commonly accepted biomarker of lipid peroxidation TBA is the reagent used to determine the amount of aldehydes or the products of lipid peroxidation. All types of aldehydes are reactive with TBA. It is aldol condensation reaction. After the reaction with different aldehydes with TBA, TBA-aldehyde adducts gives different coloured compounds. It is not an free radical assay it is a test for estimation of lipid peroxidation with its end products you can check out my publication: Ultra sensitive detection of malondialdehyde with surface enhanced Raman spectroscopy.http://download.springer.com/static/pdf/566/art%253A10.1007%252Fs00216-010-4225-3.pdf?auth66=1406078746_ae8cf38aeae9ec1b11c9493e35db32d6&ext=.pdf

A sensitive measurement of total body antioxidant level would be reflected inside of the cell. In my opinion, if you were to get a more meaningful measurement of glutathione, it would be looking at lymphocytic glutathione levels, GSH/GSSG.

Agree with Artur!