I would like to measure oxidative stress in serum samples, but my problem is the conservation of these samples, they have been stored for 7 years at -80º and were taken without caution.
you could, but shouldn't expect reliable results from this. it's hard to measure NO, for example, in such an old sample. But you could try and standardize it. good luck.
Hi! After 7 years storage I would not expect reliable results. Even -80°C is sufficient to permit ongoing biochemical reactions and ROS are intrinsically instable due to their "radical" nature (dependent on the species you are looking for). Using spin traps immediately after obtaining your serum samples would have been a way to determine ROS years later (if you had access to an NMR spectrometer). Please be not too disappointed, since it may well be possible to determine not ROS themselves, but TRACES of oxidative stress, like 8OHdG or nitrotyrosine (and numerous others). You will find a great choice of commercial ELISAs for these targets. I am no biochemist, but I assume that nitrosylated groups in complex molecules or hydroxylated DNA are stable for years under proper storage conditions.
Good luck!
HI ! storing for this much longer time i think so possibility of getting exact result is 50% or may be less than that, because free radical start deteriorating for this much longer time , if possible you can do just LPO or NO from that control human serum sample , see that its comes normal or not , then you can proceed for treated sample . gud luck
Should not expect reliable results as stated by others in the response. Antioxidants levels are likely to go down drastically and oxidants levels would most probably rise. Serum collection process itself is prone to creation of oxidant-antioxidant disturbance since you need to keep blood at room temperature or 37C followed by centrifugation to collect the serum. Both enzymatic and non-enzymatic antioxidants are likely affected by this process on the day of collection. What about after seven years. I would not expect any reliability from the data collected. It's disappointing but rigorous principles should be applied while carrying out scientific research of the highest merit.
Free radical formation and their followup reactions are dynamic phenomena. Their existence is transient in nature - very rapid is their synthesis so also same are their degradation reactions. Best analogy for for your situation is like "measuring arterial blood gases in stored samples". This especially true in case you plan to measure the free radicals themselves like HOCl, NOO, NO or their immediate reaction products that are often short lived. However if you plan measuring the final products/end products(such as Advanced glycation end products) of oxidative reactions of ROS then you may able to get some data to give you some idea, but still it may not be same as the data that is obtained by using fresh samples.
http://services.aarc.org/source/DownloadDocument/Downloaddocs/07.06.0732.pdf
Hi, Try to measure the products of oxidation, i.e. malondialdehyde (MDA) with ELISA. Best,
Hi!
Although the reliability of the result could a matter of question, you can try to measure the Total oxidant status (TOS, Erel, 2005); Total antioxidant status (TAS, Re et al., 1999) and their ratio oxidative stress index (OSI, Harma et al., 2005) which will give a picture of redox status of the sample. Apart from these you can look into the oxidative damage caused to the lipid component (MDA, HNE or TBARS as a whole) and /or protein damage (protein carbonyl, advanced oxidation protein products) as per your interest.
Good Luck!
After this much time it would be dificult to obtain reliable results. You would be spending a lot of time and money and always doubt. Good Luck!
Dear Colleague,
the worst is not the time of samples storing (7 years, without no doubt, affect the final results but that they can in any case be standardized, this is the case for example of large epidemiological studies like EPIC) but the way of storing samples. I read in another post from you (the one on d-ROM) that samples have been thawed one or more time and in different ways and without any control. If this is true there is no text or assay that can give you reliable results and any suggestions concerning the best assay to be used in serum is useless.
In any case, you work in a centre that is largely specialized in these topics and several of the scientists working there could easily give you this kind of information. I think it is not necessary I indicate who yiu can contact since most of people working in the CABD (Centro Andaluz de Biologia del Desarrollo) may help you.
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