Dear Charlotte,

The usual methods for measuring antioxidant activity (AA) of plasma (notably FRAP) only assess the AA of low molecular weight compounds diluted in plasma which possess a certain property (redox potential in the case of FRAP). Enzymes involved in the antioxidant system do not operate by these ways, so they are not measured by such assays. However, you are correct to say that the AA of plasma is not entirely due to non-enzymatic antioxidants, but rather to the sum of both enzymatic and non-enzymatic antioxidants. In fact, many studies indicate that the contrubution of enzymes to the human body's antioxidant defense is much higher than that of non-enzymatic antioxidants, such as phenolic compounds.

I hope this could be of help.

Kind regards,

Daniel

Dear Charlotte,

it really depends on the biochemistry of the assay you use. Usually many of these assays only quantify the "activity" of non-enzymatic antioxidants that occur in your sample by letting them to react in vitro with an oxidant (radical or not radical). So, while the body antioxidant defences of course include many different kinds of molecules, the so-called tac assays usually quantify only the non-enzymatic ones that occur in your biological matrix (eg, plasma, emolysate, tissue homogenate).

Hope this helps

David

there are non-enzymatic antioxidant defense mechanisms involving different chemical groups, e.g. vitamins, carotenoids, amino acids and peptides which are established in various cellular structures to deal with anti-oxidative effects.The all antioxidant factors of the body of an organism, either intracellular enzymes or antioxidant nutritional compounds (non-enzymatic factors), are called total antioxidant capacity.

also,In a study conducted by Winston et al. (1998), it was revealed that glutathione, ascorbic acid (vitamin C), uric acid and vitamin E compose 70% of the total antioxidant (Winston et al., 1998).

I think that major researchers have been examining the antioxidant activities of plasma which proteins are removed with kinds of acids, prior to the measurements.

This means that almost all blood proteins and enzymes can be removed from the tested samples, then the activities of the sample should be derived from non-enzymatic constituents in the plasma.

I recommend you to see details of methods of antioxidant measurements or papers. If organic solvents are used in the measurements as the reaction solvent, proteins can be precipitates before measuring the activities.

Hello, yes, but removing proteins from the sample means also to remove protein thiols, which are an important non-enzymatic antioxidant component of plasma. Again, it depends on the assay. For example, I have used several times the OXY assay which is based on an in vitro reaction between HOCl and antioxidants in your matrix. HOCl is not a substrate for classic antioxidant enzymes like SOD, GPX or CAT, so their contribution to antioxidant capacity, if any, is negligible.

Cheers

David

Thanks all for your contributions! @Junichiro Wakamatsu: I assumed here that no particular treatment was done to remove proteins, as this is not done in many papers I came across.

From your answers and what I know about the systems and assays involved, here are the main conclusions I could draw, and the main issues that remain:

First, assays involving a direct redox reaction between antioxidants and an oxidant probe (including FRAP) basically measure the redox potential of antioxidant compounds, assuming that this is the main determinant of their activity. This is definitively not true for enzymes, but could also not hold for some non-enzymatic antioxidants.

In assays that measure the antioxidants reaction with peroxyl radicals from lipid chains or azo compounds, superoxide and hydroperoxide would not be produced in significant amounts, so the SOD and catalase enzymes would not be involved in the reaction. However, some gluthatione peroxidases have an antioxidant activity toward lipid peroxyl radicals. Moreover, are all non-enzymatic antioxidants able to react with peroxyl or alkoxyl radicals in these settings, or are these assays also limited in the range of NE antioxidants they measure?

To summarize, most assays are not expected to produce much free superoxide or hydrogen peroxide, and SOD and catalase are thus not expected to react. It seems to me though that the question remains of (1) the contribution of GPx to some assays that involve lipid peroxyls (maybe the GSH levels are limiting to GPx activity in the plasma, unlike their high concentration within cells?), and (2) the range of non-enzymatic antioxidants actually measured by a given assay.

Eventually, although the OXY test measures antioxidant activity toward HOCl, couldn't HOCl also increase the levels of other ROS that would then react with antioxidant enzymes? I was mainly thinking of H2O2, but do not know whether H2O+HOCl->H2O2+HCl would occur in biological samples. Most antioxidant enzymes are at least partially inactivated by HOCl, but the HOCl concentration and the reaction time might not be enough in the assay.

Considering (at least for the OXY) the reaction time, amout of HOCl used for the reaction, low concentration of H2O2 in plasma and concentration of plasma antioxidant enzymes it is basically impossible to have a significant contribution of other oxidants or enzymes. In zebra finches, I tried to check for potential correlations between plasma OXY and plasma GPX (or rbcOXY and rbcGPX), but I only found no correlations or small significant but negative correlations (J Exp Biol 2011 214, 1148-1152). The real issue here is that these assays have many limits, first of all if this in vitro antioxidant capacity really reflects that in vivo. But they may have some diagnostic value.

Dear Charlotte,

Your question makes definitely sense, in strict meaning, as TAC comprises both enzymatic and non-enzymatic moieties contributing to it by definition. Nonetheless, I would like to rise a more general question, about TAC assays on plasma or serum: do they provide a really useful picture on the overall redox status (or, simply, about the TAC level) of the body? In my view, the point is questionable in view of two considerations: i) enzymatic and non-enzymatic machinery in charge of redox equilibrium maintenance is located inside cells; ii) no clear evidence has been provided (to my knowledge) in the literature about any linear relationship between plasma TAC and intracellular TAC. 

Based on these premise, I would suggest to measure TAC (including the enzymatic component) on blood red cells. This can be carried out  by measuring single analytes by reliable methods, with (for metabolites) or without (for enzymes) deproteinisation. The contribution of metabolites to TAC may be alternatively quantified by a new method based on a redox indicator (see attached file).

Best regards,

GL

Hi Charlotte,

Just saw this old thread but thought I'd respond because I have some relevant unpublished data. I can only speak to the TEAC assay (sometimes called TAS or TAC), described in my 2007 paper "Measuring antioxidants in wild birds." That paper describes the time series of the spectrophotometric readings: a flat low initial absorbance, followed by an increase (initially linear, then plateauing). I recommended not using the commercial method of just taking the reading at 3 minutes because this confounds the timing of the initial increase, and the rate of increase/plateauing. 

What I did not say in that article is that we have good reason to think the initial timing (length of the flat phase) is due to non-enzymatic antioxdants, whereas the rate of increase is likely enzymatic, though we do not have firm proof. All the controls use standard non-enzymatic antioxidants followed by a linear increase (no plateau). In contrast, when we tried to run samples on homogenized chicken tissues, we had no initial delay but a very quick plateau without much linear increase. We got similar results when blood samples had too much hemolysis. My conclusion was that enzymatic antioxidants, which are high within cells but not in plasma/serum, contribute to the plateauing and the slope of the initial linear increase.

This was all 10-12 years ago, so my memory is a bit fuzzy, but I think the interpretation as non-enzymatic is basically right (though with perhaps a few nuances) as long as one uses the method I proposed of using the time to initial increase rather than absorbance at 3 minutes. I would expect similar properties for some of the other assays, but would be cautious in interpretation.

All the best,

Alan