I'm trying to run some experiments dealing with in vitro oxidative damage, using commercial preparations of blood plasma as a representative biological medium. The assay I'm using is the ferric reducing ability of plasma (FRAP) assay. I'm trying to establish a positive control for damaged plasma. In other words, I don't want a negative control such as plain methanol or something similar i.e. the complete absence of antioxidants; I want to use plasma that had an antioxidant potential to begin with, but that I abolished or damaged using some treatment.
Originally I was trying to use hydrogen peroxide in order to oxidize the antioxidants in plasma, but today we discovered something very curious; there is a distinct positive correlation between increased concentrations of H2O2 that are being "spiked" into blood plasma, and apparent increases in FRAP from a kinetic standpoint. It sounds confusing and completely contrary to what you'd expect, but the higher the concentration of H2O2 you use in plasma, the faster and more intensely the purple colour of the (TPTZ)2-Fe2+ complex develops at 585 nm.
I was absolutely perplexed as to how this could possibly be at first, but I suppose it's due to one of the following (or a combination of the two):
a) Some sort of Fenton reaction/Haber-Weiss cycle of peroxide-treated plasma is affecting both the iron in plasma and the FeCl3 of the FRAP reagent, and making it seem as though blood plasma spiked with H2O2 has way more antioxidant potential than untreated plasma. I'm not quite sure of radical and/or redox mechanisms of this (I would have thought the iron might "cycle" between Fe3+ and Fe2+ but not remain "locked" at Fe2+), but I can't ignore the fact that increasing [H2O2] positively correlates with increasing kinetic development of the coloured complex at 585 nm;
b) The longer H2O2 is left to incubate with blood plasma, the more it is damaging plasma proteins, and causing potential release of iron from those proteins (e.g.transferrin). That said, those proteins will still bind Fe3+, not Fe2+, so the question remains of how Fe3+ is being reduced.
Whether the case is a) or b) or something else is really neither here nor there, truthfully. The *ultimate* question of all this is: is there a way to "damage" the antioxidant potential of plasma such as to establish a positive control for decreased FRAP, while having the results of the assay actually make sense?
Fenton reaction will not be limited by whether the ferrous ion is free.
try looking at these links maybe you can help!
http://www.google.it/url?sa=t&rct=j&q=&esrc=s&source=web&cd=5&cad=rja&uact=8&ved=0CGAQFjAE&url=http%3A%2F%2Fwww.ijpbs.com%2Fijpbsadmin%2Fupload%2Fijpbs_50cdc36950ea3.pdf&ei=G_19U6KlManZ4QSJqIHYBQ&usg=AFQjCNHwERoUC-QEmntDbcfFKXIELwmRfA&sig2=DdPQlbpAnRuhtN0KruEAmQ
http://www.google.it/url?sa=t&rct=j&q=&esrc=s&source=web&cd=6&cad=rja&uact=8&ved=0CG0QFjAF&url=http%3A%2F%2Fxa.yimg.com%2Fkq%2Fgroups%2F9534928%2F1879535612%2Fname%2FThe%2BFerric%2BReducing%2BAbility%2Bof%2BPlasma%2B(FRAP)%2Bas%2Ba%2BMeasure%2Bof%2B%25E2%2580%259CAntioxidant%2BPower%25E2%2580%259D.pdf&ei=G_19U6KlManZ4QSJqIHYBQ&usg=AFQjCNHGuU76VB5A2D0ZJqU485Zy_QivEQ&sig2=7i3A6--Zykjuyum_8LQeyw
Go through following paper..
Http://www.clinchem.org/content/44/6/1309.full
Please check this link;
Ferric Reducing Ability of Plasma with Lipid Peroxidation in type 2 diabetes PJ Hisalkar*1, AB Patne1, AC Karnik 1, MM Fawade 2, SS Mumbare 3 1Dept of Biochemistry, ACPM Medical College & Hospital, Dhule 2 Dept of Biochemistry, Dr BAMU Aurangabad 3Dept of Community Medicine, PSM, Dr.Vasantrao Pawar Med. Col. Hosp. & Research Centre, Nasik
http://www.ijpbs.com/ijpbsadmin/upload/ijpbs_50cdc36950ea3.pdf
Hi all,
Thanks for the great responses so far. Having looked through all the links you've generously supplied, I'm not really seeing much of a positive control for FRAP depletion though. The best I've seen so far (and "best" is a term I use loosely) is plasma that has been sitting in the freezer for a few months and is "aged."
Is there a semi-instantaneous treatment that can simulate oxidative stress in plasma, such that FRAP levels will be lowered?
Thanks again
Question: : Is there a way to "damage" the antioxidant potential of plasma such as to establish a positive control for decreased FRAP, while having the results of the assay actually make sense?
Answer: It will be difficult to use the positive contral for FRAP assay. How is about using the control plasma added with any of standard polyphenolic compounds.?
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