1. Principle

Several compound are able in the plasma to “buffer” the oxidant potential of oxygen reactive species. Virtually, any compounds, either endogenous (i. e. GSH, thiols, proteins, bilirubin, uric acid, cholesterol, etc.) or “endogenous” (i. e. ß - carotene, ascorbate, vitamin E, etc.) able to give electrons is able also to block the potential damage of a free radical, because the reactivity of a free radical is just due to the lack of electrons. Obviously, any injury to such “plasma/serum barrier to oxidation” can result in oxidative tissue damage.
The OXY–Adsorbent test evaluates the plasma/serum ability to oppose the massive oxidative action of a hypochlorous acid (HClO) solution. In the OXY-Adsorbent test HClO was selected among other oxidant agents because it is not only a powerful but also a physiological antioxidant. Therefore it is able to reproduce in vitro some situations which occur in vivo.
Briefly, in the OXY-Adsorbent test the efficacy of the blood antioxidant system is monitored indirectly by measuring the excess of HClO in serum (released as radical).As unreacted radicals of HClO react with a correctly buffered chromogenic substrate (N,N-diethylparaphenylendiamine), a colored complex develops that can be measured photometrically, presenting a maximum peak of absorbance at 505 or 546 nm. The concentration of the colored complex is directly proportional to the concentration of HClO and indirectly related to the antioxidant capacity.

2. Composition of kits
A typical kit of OXY-Adsorbent test contains, basically, an oxidant solution (R1 reagent), a chromogenic mixture (R2 reagent) and a calibrator (R3 reagent) (table1). The analysis can be carried out either by means a dedicated instrumentation (i. e. FREE system) or a normal photometer.
To calibrate the analytical instrumentation a lyophilized control serum with assigned value is available.

3. Analytical procedure
The OXY-Asdsorbent test can be performed on fresh serum or plasma according to the following working conditions: wavelength 505 or 546 nm, optical path 1 cm, and room temperature. The analysis can be carried only according to the fixed time mode.
To carry out OXY-Adsorbent test, 10 µL of sample (fresh serum or plasma), previously diluted 1:100 with distilled water, are added and mixed with 1 mL of oxidant solution (R1). After 10 min of incubation, at 37°C, 10 µL of chromogenic mixture (R2) are added. For each series of assays, a standard with assigned value, previously diluted 1:100 with distilled water as for the samples, and a blank reagent, obtained by replacing serum with distilled water, must be included. The absorbances must be measured immediately at 505 nm or 546 nm. The absorbance value of the reagent blank is subtracted from those of the standard and the samples. The antioxidant capacity, expressed as µmoles HClO/mL of sample, must be calculated by this formula (table 2).

4. Results interpretation
Normally, 1 mL of human plasma is able to adsorb at least 350 µ mol of hypochlorous acid. Decreased values directly correlates to the injury severity of “the plasma barrier to oxidation” (table 3). In fact, when the “excess” of radicals of hypochlorous acid after massive oxidation is high, the plasma barrier is obviously reduced, and vice versa.

5. Clinical studies
The OXY-Adsorbent test was shown to be a very reliable test to evaluate antioxidant capacity of human serum in several diseases, thus integrating the contribute of d-ROMs test in the optic of the global assessment of oxidative stress. Indeed, OXY-Adsorbent test should be performed every time d-ROMs test value is high and a monitoring of antioxidant therapy is required.
Among the published trials, Carratelli and Coll. (Int J Clin Pharm Res, 2001) have demonstrated that children with Down syndrome have, compared to controls, significantly higher d-ROMs test values and lower OXY-Adsorbent test values (p<0.05, patients vs controls, see figure 1). It is probable that oxidative stress observed in such population of children suffering by Down syndrome is the result of a reduced efficacy of plasma antioxidant barrier (low values of OXY-Adsorbent test) that appears to be not able to eliminate the excess of reactive species (high values of d-ROMs test).
Similar results were found also by Ippolito and Coll. (Biochimica Clinica, 2001) in myelodysplastic patients, compared to controls.
Trotti e Coll. (Haematologica, 2001), on the contrary, have found that heavy drinkers (without severe liver disease) have significantly higher value of d-ROMs test compared to controls (no drinkers). However, between the two group of subjects enrolled in this trial a statistically significant difference regarding to the values of OXY-Adsorbent test was not found. These findings suggest that in heavy drinkers the “signs” of oxidative damage (high d-ROMs test values) can co-exist with an antioxidant barrier practically normal (normal OXY-Adsorbent test values). Therefore, in this population oxidative stress can result from an exaggerate reactive species production rather than an impairment of the antioxidant barrier. Similar results were found also by Iorio e coll. (2001) in a preliminary report on obese patients compared to controls. Also in these patients, in fact, d-ROMs test provided high values while OXY-Adsorbent was not significantly low.
The results of above mentioned trials indicate that OXY-Adsorbent test provides very useful information in the assessment of oxidative stress. However it should underline that high value of d-ROMs test not necessary must co-exist with low values of OXY-Adsorbent test.
Finally, it must be remembered that beside clinical applications, OXY-Adsorbent test, by means of appropriate technical fittings, can be carried out also on vegetal extracts in aqueous phase (i. e. fruit juices, tomato juice, wines, etc.). This test, therefore, is very useful to assess antioxidant activity of any water-soluble compounds virtually provided of anti-radical activity.