Hello everybody,
I am currently searching for some oxidative stress markers, which could be measured in frozen serum, after lets say - about 6 month storage in -80 Celsius temperature.
That is because group of my patients would be relatively small and I need some time to qualify patients for the study.
Any good ideas or experiences you can share with me?
Please have a look at the publication in the attachment. Maybe it can be helpful to you. Some of the authors are members of RG so you can ask them for sending you a pdf copy.
Regards, Christian
Article Evaluation of reactive oxygen metabolites in frozen serum sa...
A study was designed to determine the effect of storage at -30 degrees C and -80 degrees C for two years on free radical activity. The free radical activity was analyzed by measuring circulating hydroperoxides in a pool of sera at baseline and after one day, one week, one month and 25 months of storage, using a photometric method (d-ROMs test). Measurements were performed in aliquots thawed only once at each time point and in aliquots frozen and thawed repeatedly over the study period. After two years a small but statistically significant 4% decrease in the hydroperoxide concentration was observed, which was substantially unaffected by storage temperatures and repeated freeze-thaw cycles. In conclusion, the d-ROMs test is a simple method suitable to evaluate the free radical activity in frozen serum samples after long-term storage. (Cavarelli et al., 2004)
Oxidative stress is a very complex process, which is highly dependent on the metabolic phenotype of an organism (i.e. genetic diversity), type of organ, and place in the cell. Because there are many radicals, which have different mechanism of formation and action, there many types of markers. Loss of activity of some enzymes due to damage to the 4Fe-4S claster in the active center, is the marker for the direct effect of superoxide radical. Accumulation of 4-Hydroxynonenal and its adducts with proteins, for example, \is a marker for increased oxidative stress, whereas accumulation of adducts of isoketals with lysine-containing proteins or phosphatidylethanolamines is a sign of the isoprostane pathway of lipid peroxidation? Whereas accumulation of nitrated tyrosine-containing proteins is a marker for increased production of peroxynitrite or peroxynitrate, etc.
Thus, you have to know more about radicals to plan your experiments. But you have to remember two important things. First, measurements of radicals and the products of their action is a complicated matter and require expensive equipments and, most importantly, knowledge and skills in their use and interpretation of the data. It is the Nature’s Law: Important information is expensive. The second error, most common among young researchers and medics, is the hope that “we take a sample of blood, tissue or else, freeze, and later determine whatever we want”. Basically that might work, under condition that you will keep your samples in liquid nitrogen, which is a very expensive procedure. And you can defrost your sample only once. The dangers of the storage of biological samples at -20C are described in the papers from Vanderbilt University (see references below) and in my book “Practical mitochodriology” I the section about submitochondrial particles (you can find it on my page in Research Gate).
References.
Roberts L.J., 2nd, T.J. Montine, W.R. Markesbery, A.R. Tapper, P. Hardy, S. Chemtob, W.D. Dettbarn and J.D. Morrow. (1998) Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid. J. Biol. Chem. 273, 13605-13612
Roberts L.J., 2nd, Fessel J.P. (2004) The biochemistry of the isoprostane, neuroprostane, and isofuran pathways of lipid peroxidation. Chem. Phys. Lipids. 128(1-2), 173-186
Roberts L.J. and J.D. Morrow. (2000) Measurement of F(2)-isoprostanes as an index of oxidative stress in vivo. Free Radic. Biol. Med. 28(4), 505-513
- Badges
- Science method