Reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) are produced as a consequence of normal aerobic metabolism in animal species [24–26]. These "free radicals" are removed and/or inactivated in vivo by a battery of antioxidants [4, 24–27]. A biological antioxidant is defined as a substance, which, at low concentrations compared to that of the oxidisable substrate, significantly delays or prevents this oxidation [6, 27, 28]. Individual members of the antioxidant defense team are employed to prevent the generation of free ROS and RNS, to destroy potential antioxidants and to scavenge ROS and RNS. However, the relative sufficiency of the organism antioxidant defenses is critical in the development of oxidative stress in patients affected by a number of diseases, including HIV infections [29, 30], neurodegeneration , diabetes [3, 32–34], angina [35–38], certain forms of cancer [39–47], and probably ageing [48–50]. These diseases are characterized by an overproduction of free radicals, i.e. when the antioxidant defense of an organism is overwhelmed or are established when a deficit of defenses of the organism against oxidation occurs.
The primary defense against oxidative stress in extracellular fluids results from a number of low molecular weight antioxidant molecules either water – (ex. ascorbic acid) or lipid-soluble (ex. Vitamin E). These antioxidants can also be generated during normal metabolism (ex. uric acid, bilirubin, albumin, thiols) or introduced in the body by the consumption of dietary products rich in antioxidants (olive oil, fruits and vegetables, tea, wine, etc) . The sum of endogenous and food-derived antioxidants represents the total antioxidant activity of the extracellular fluid. In addition, the levels of these antioxidants are suitable not only as a protection against oxidation, but could also reflect their consumption during acute oxidative stress states. The cooperation among different antioxidants provides a greater protection against attack by reactive oxygen or nitrogen radicals, than any single compound alone. Thus, the overall antioxidant capacity may give more relevant biological information compared to that obtained by the measurement of individual parameters, as it considers the cumulative effect of all antioxidants present in plasma and body fluids . A theory has recently be proposed, taking into account the redox potentials of exogenous and endogenous antioxidants, and the construction of a chained reaction, in which a given antioxidant, after oxidation is regenerated through a number of reactions involving a number of other, more potent antioxidants. Through this cascade, interactions among the lipid and the aqueous phases could be established .
A great variety of methods have been proposed for the assay of total antioxidant activity or capacity of serum or plasma [reviewed extensively and critically in [6, 51]]. In these reviews a clear distinction among antioxidant activity and capacity is made: Antioxidant activity corresponds to the rate constant of a single antioxidant against a given free radical. Antioxidant capacity, on the other hand, is the measure of moles of a given free radical scavenged by a test solution, independently of the capacity of any one antioxidant present in the mixture . Therefore, for plasma, being a heterogeneous solution of diverse antioxidants, the antioxidant status is better reflected by antioxidant capacity rather than activity. This capacity is a combination of all the redox chain antioxidants, including different analytes such as thiol bearing proteins, and uric acid. Therefore, the plasma antioxidant capacity is rather a concept than a simple analytical determination. Indeed, an increase of the antioxidant capacity of plasma indicates absorption of antioxidants and an improved in vivo antioxidant status , or an adaptation mechanism to an increased oxidative stress. Nevertheless, due to the participation of diverse metabolites (see Figures 6 and 7) to the antioxidant capacity of human plasma, its increase may not be necessarily a desirable condition. Indeed, in some cases, such as renal failure (uric acid), icteric status (bilirubin), hepatic damage (hypoalbuminemia) the variation of several metabolites falsely modifies the plasma antioxidant capacity, a situation returning to normal values after correction of the underlying disease .
As derived from the definition of antioxidant capacity, and the heterogeneity of antioxidant substances in human plasma, all methods used for its determination are by definition indirect . The crocin bleaching method, used in the present paper, initially described by Tubaro et al , and Lusignoli et al  uses crocin oxidation by peroxyl radicals produced by ABAP. By comparing the inhibition of bleaching (oxidation) of crocin by plasma, to an artificial antioxidant (Trolox C), either kinetically , or at end point , a standard antioxidant capacity of plasma can be derived, expressed as Trolox equivalent. As discussed by Prior and Cao , a serious problem of the crocin method is the lag time phase, when lipids and proteins act as antioxidants, a result not encountered in the modification proposed here (Figure 7), at analyte concentrations exceeding by far the reference values in human plasma. In addition, concerning ascorbic acid (that the previous kinetic method provides values exceeding all other reported, 7.7 Trolox equivalents) was not a problem in the current assay. Indeed, ascorbic acid accounted (on a molar basis) only for 1.22 Trolox equivalents. Compared to another commercialized antioxidant capacity determination (Total Antioxidant Status by Randox) (see Figure 4), a significant linear correlation was observed, while TAC assay tends to assay lower AC by 0.5 mmol/L, expressed as Trolox equivalents. The TAS assay is based on the TEAC (Trolox Equivalent Antioxidant Capacity) method, reported by Miller and Rice-Evans [21–23]. It is based on the inhibition by antioxidants of the absorbance of the radical cation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) formed by the interaction of ABTS with ferrylmyoglobin radical species. This, also indirect, method gave similar reference values as the TAC assay, on 44 healthy blood donors (TAS: 1.209 ± 0.005 mmol/L; TAC: 1.175 ± 0.007 mmol/L of Trolox equivalents), measured serially on the same autoanalyzer.
As discussed elegantly by Prior et al , different metabolites interfere with all indirect antioxidant capacity methods. These endogenous analytes include uric acid, ascorbate, albumin, bilirubin and lipoproteins. During the validation of the TAC assay we performed analysis of the above metabolites on the antioxidant capacity. We have found that uric acid, bilirubin, ascorbate and lipoproteins accounted for 0.11 mmol/mg, 0.14 mmol/mg, 0.07 mmol/mg, and 0.18 mmol/100 mg respectively. Taking into account the normal concentrations of these analytes, it was concluded that about 1 mmol/L (i.e. about 85% of the TAC) is due to endogenous analytes, and only 15% of the observed TAC might be due to exogenously provided antioxidants. Of course, as our reference subjects were blood donors, we could not have precise evaluation of their dietary and smoking habits. Indeed, it is well established that smoking habits reduce the TAC of human plasma, a reduction which is reversed after stopping smoking . Non-smoking nuns, following a diet rich in antioxidant substances, increase dramatically their TAC of plasma, demonstrating the importance of dietary antioxidants (Figure 8).