Question
Oxidative stress analysis
Can isolated red blood cells be used for oxidative stress analysis following incubation with an oxidation compound? (in-vitro oxidative stress analysis in red blood cells)
All Answers (31)
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Yes and also liver and brain homogenates can also be used -
Yes, u may need to prepare the erythrocyte haemolysate and perform tests similar to polymerization -
Dear Natalia,
Yes, you can use red blood cells for oxidative stress analysis. A large panel of assays would be useful to understand this metabolism. Please find few enzymatic activities and metabolites that BioQuanta's team can measure, at once, on the same sample :
- SUPEROXYDE DISMUTASE Cu/Zn (SOD Cu/Zn)
- SUPEROXYDE DISMUTASE Mn (SOD Mn)
- GLUTATHION Peroxidase TOTAL GPX
- CATALASE
- GLUTATHION Reduced and oxidised (GSH & GSSG)
- GLUTATHION Reductase (GR)
- GLUTATHION Synthase : γ-Glutamylcysteine glycine ligase + γ-Glutamylcysteine synthetase
- GLUCOSE 6 Phosphate Deshydrogenase G6PDH
- MALONALDEHYDE (MDA)
Does my answer help you ?
Best regards,
Romain -
you can use red blood cells for oxidative stress analysis .. in vitro... read my article....
Mansour, S.A.; A.H. Mossa and T.M. Heikal (2009). Effect of methomyl on lipid peroxidation and antioxidant enzymes in rat erythrocytes: in vitro studies. Toxicology Industrial Health. 25 (8): 557-563. -
To romain: mn-sod in rbcs? -
I have no experience with oxidative stress in isolated red blood cells, but I think that you can do it. You can perform the studies using H2O2, tert-butylhydroperoxyde (as external oxidative stress agents). After treatment, incubate the cells with diclorodihydrofluorescein-diacetate (H2DFCDA) and you should perform FACS analysis or fluorescencece measurements. -
What sort of stress analysis are you looking for? What is being analyzed? If this idea is to detect reactive oxygen species, there are probes for this. I cannot think of any application where I would use red cells unless the object was to study hemolysis.
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Yes, you can -
Yes, you can do and use probes detect NO or whatever other ROS. -
Thank so much Dr.Abdeltawab Mossa.
but for me I am working in oxidation stress( transportation stress).
I use plasma for analysis of oxidative stress analysis. -
I have no experience in oxidative stress in isolated red cells, but I think it would be difficult to study since they first need to know if the red cells have mitochondria and smooth endoplasmic reticulum, since most of the enzymes are located in these organelles and second whether the induction of enzymes by oxidative stress is transcriptional postraducional or traducional; in the case of enzymes that are induced transcriptionally (hemeoxigenase 1) this would be difficult because red cells have no nucleus, and finally in erythrocytes the iron of the hemoglobin would favor the reaction of Fenton and cause many reactive oxygen species in particular those derived from lipid peroxidation which cause lysis.
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You can use the isolated red blood cells following incubation with an oxidant compound to study oxidative stress related parameters. Be carefull at the incubation buffer and at the concentration of the compound of interest in order to avoid hemolysis. Usually we are measuring on erythrocytes antioxidant enzymes activity , free and total thiols groups, MDA. The assays are not expensive and quite easy to adapt on an automatic analyser(we are using for example COBAS) or on a microplate reader .
You have also to be carefull at the temperature you are keeping the samples after you decide to hemolyse the cells and to do your analyses.We are keeping the samples on ice.I have worked a lot oxidative stress related parameters on erythrocites so If you need more information for speciffic assays you decide to do feel free to contact me and I will answer with pleasure -
oxidative stress is associated with oxidative phosphorylation prokaryotes lack mitochondria so this process takes place accross the plasma membrane. My suggestion would be read Abdeltawab Mossa paper and use Romain Guidon strategy to see if red blood cells have oxidative stress defenses ( am assuming you are looking for this), but be careful with concentrations you use to stress the cells i would suggest very low concentrations of hydrogen peroxide or menadione (superoxide anion donor), meaning micro molar concentrations.
Hope this helps -
You can determined lipoperoxidation to quantify membrane lipid alteration. -
Yes, you can. You can detect oxidative modifaied proteins (total), produts of lipid peroxidation, activity of antioxidat enzymes (SOD, catalase, GPO, G-S-TF). -
Just look at RBC hemolysis- oxidatively damaged RBC will have an increased RBC hemolysis. -
Yes, you can. You can detect stress enzymes activity and stress product. As well as micronucei assay.
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Yes, you can detect oxidative stress in erythrocytes, tissues, plasma ..etc.
My articles
1. Mansour S.A. and A.H. Mossa (2010). Adverse effects of lactational exposure to chlorpyrifos in suckling rats. Human & Experimental Toxicology. 92 (2): 77-92.
2. Mansour S.A. and A.H. Mossa (2010). Oxidative damage, biochemical and histopathological alteration in rat exposed to chlorpyrifos and the role of zinc as antioxidant. Pest. Biochem. Physiol. 96: 14-23.
Also,
Methods for assessment of free radical activity in biology
Many approaches allow evaluation and demonstration of the participation of ROS in biochemical events (Fig.). However, the choice of which indicator(s) of free radical activity to study is complex. In view of the lack of any ‘standard’ assay of free radical activity we have always attempted to follow the following multiple approaches where practical:
1. Measurement of endogenous antioxidant levels.
2. Measurement of indirect indicators of free radical activity, e.g. products of lipid peroxidation, products of DNA oxidation, products of protein oxidation.
3. Measurement of a direct indicator of free radical activity, i.e. electron spins resonance (ESR) studies. -
Yes you can assess the oxidative stress parameter such as SOD, catalase, lipid peroxidation, and even antioxidants like vitamins A, E, C glutathione rdeuctase, glutathione peroxidase in the hemolysate of RBC. But take care for the pH of the buffers and temperature at which you are analysing your samples. -
Yes, you can analyse oxidative stress in blood cells. The main precautions to prepare incubation buffer. It should be prepare carefully with accurate pH.
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As man people already commented RBC can represent a good model to study oxidative stress parameters both in vivo and ex vivo.
In particular the compartment you could look at are isolated membranes after lysis (ghosts) where you can assess oxidatin of lipid components but also phisical-chemical changes in the properties of the membrane using fluorescence methods (rigidity, fluidity of membranes); In hemolisate you can evaluate low molecaluar weight antioxidants and enzymatic antioxidants such as SOD, CAT and GPx, but also the oxidative state of hemoglobin is an interesting endpoint (oxi and meta Hb levels) in particular if you study it dinamically following an incubation with prooxidant agent to induce hemolisis where you can study in parallel hemolyisis and hemoglobin state. It is true that pH is crucial since it could destabilize Hb oxidation and promote ROS production altering your experimental setup.
Another dinamic test that could be influenced and interesting to correlate with oxidative stress parameters that you are going to consider is osmotic fragility of RBC.
All these aspects are valid if you are into upper vertebrate RBC, but if you have a choice and wish to move into lower vertebrates (we have an experience with fish erythrocytes for example)...this is another world since they have nucleus and mitochondria so you can study also oxidative damage to DNA and mitochondrial functionality as well as expression of genes since they are both metabolically and transcriptionally active. Check our work on fish RBCs if you are interested (Tiano L. and Falcioni G.) and let me know if you have questions. -
Thanks for all your comments!!
Indid this was very helpful.
Cheers,
Natalia -
after an oxidative treatment performed on red blood cells , to evaluate the oxidation you can use these two kit (produced by (DIACRON INTERNATIONAL; Grosseto, Italy) ) LIPOCELL and GSH .
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you can measure the concentration of lipid peroxidation product (MDA-malone dialdehyde). -
• Peripheral blood processing
Collected blood was centrifuged at 2000-3000g for 15 min and plasma was separated. Then, the plasma was deproteinised with 25% trichloroacetic acid by continuous mixing for 5 min and centrifugation at 2000g for 15 min.
• Erythrocyte Processing
The erythrocyte pellet was washed three times with saline and lysed by Ivanov (1999) with minor modifications. In brief: the erythrocyte pellet was washed three times with saline, and the cell suspension was diluted with cold water to lyse the erythrocytes. To 0.2 mL lysate, 1.8 mL water and ethanol/chloroform (3:5/v:v) were then added to precipitate haemoglobin. The tubes were shaken vigorously for 5 min and centrifuged at 2500g for 20 min. The supernatants were used for determine enzyme activity.
• Determination of products of lipid peroxidation
The deproteinised plasma was used for determining lipid peroxidation products. By spectrophotometry the total amount of lipid peroxidation products in plasma was assayed using the thiobarbituric acid reactive substance (TBARS) method, measuring spectrophotometrically MDA at 532 nm (Plaсer et al., 1966). Results were expressed as M.
• Determination of superoxide dismutase activity
Erythrocyte lysates were assayed for Cu/Zn SOD activity by method of Sun et al. (Sun et al., 1988) with minor modifications: Briefly, the xanthine/xanthine oxidase system was used to generate the superoxide anion (O2.–)x. This anion reduced nitroblue tetrazolium (NBT) to formazan, which was monitored at 560 nm. SOD in the sample removes the O2.– and inhibits the reduction. The level of this reduction is used as a measure of SOD activity. One unit of enzymatic activity is defined as the amount of enzyme causing 50% inhibition of the reduction of NBT to formazan. Results were expressed as units per g haemoglobin (U/gHb).
• Determination of catalase activity
CAT activity was assessed in the erythrocyte lysates by the method described by Beers and Sizer (19). Hydrogen peroxide was used as a substrate and the decrease in H2O2 concentration at 22oC in phosphate buffer (pH 7) was followed spectroscopically at 240 nm. One unit of CAT activity is defined as the amount of enzyme that degrades 1M H2O2 per min. Results are presented as units per g haemoglobin (U/gHb).
• Haemoglobin concentrations
Haemoglobin concentrations of lysates were analysed by cyanmethaemoglobin method (Mahoney, et al., 1993). -
Thanks for your help and for the valuable information.
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thank for this information. I am now working in biomarker of oxidative stress.
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spicaly on MDA, SOD and TAC. Add to Zn and Cu.
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Yes. Oxidation parameters can be assayed in RBCs. Similarly, they can be assayed from tissue/organ homogenates.
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Thanks for all your comments. thank so much for all.
Popular Answers
My articles
1. Mansour S.A. and A.H. Mossa (2010). Adverse effects of lactational exposure to chlorpyrifos in suckling rats. Human & Experimental Toxicology. 92 (2): 77-92.
2. Mansour S.A. and A.H. Mossa (2010). Oxidative damage, biochemical and histopathological alteration in rat exposed to chlorpyrifos and the role of zinc as antioxidant. Pest. Biochem. Physiol. 96: 14-23.
Also,
Methods for assessment of free radical activity in biology
Many approaches allow evaluation and demonstration of the participation of ROS in biochemical events (Fig.). However, the choice of which indicator(s) of free radical activity to study is complex. In view of the lack of any ‘standard’ assay of free radical activity we have always attempted to follow the following multiple approaches where practical:
1. Measurement of endogenous antioxidant levels.
2. Measurement of indirect indicators of free radical activity, e.g. products of lipid peroxidation, products of DNA oxidation, products of protein oxidation.
3. Measurement of a direct indicator of free radical activity, i.e. electron spins resonance (ESR) studies.