A new procedure for the Antioxidant capacity dpph assessment in small samples.

The antioxidant capacity of a substance is its ability to prevent or slow down oxidation. This reaction is a transfer of electrons. The molecule catching electrons is the oxidative agent and the molecule giving electrons is the reducing agent. Oxidative stress is toxic for the cells, and it can be limited by different pathways: the inhibition of enzymes responsible of the production of oxidative species, the synthesis, activation or stabilization of antioxidant enzymes, and finally by direct scavenging of radical species. In living organisms, especially in association to the respiration process, free radicals are commonly produced. Antioxidant are naturally present to prevent damages, and therefore the antioxidant capacity of a substance is a key element to evaluate. Several methods can be employed to assess total antioxidant capacity in a liquid, one of the most famous requires the use of Di-phenyl-picryl-hydrazyl (DPPH). Brand & Williams developed in 1995 the method of assessment of antioxidant capacity based on the color change of DPPH according to its oxidation status. The oxidized DPPH is a radical, with dark purple color and in contact with antioxidant species, it is reduced and turns to yellow-green color. The amount of oxidized DPPH can be quantified at 517nm spectrophotometrically. Using a known dose of antioxidant molecule, such as Trolox, a standard curve of antioxidant can be built. The antioxidant capacity of any sample can be assessed measuring the oxidized DPPH, in Trolox equivalents. The main limitation of this method are (i) the important volume of sample required and (ii) the interference of molecules of fat present in biological fluids. A new protocol with adaptations has been developed in order to obtain efficient and reproducible antioxidant capacity measurement in very small biological samples, with a required volume 20 times smaller than in the original method. We used it for antioxidant determination of colostrum, which available quantity is very limited, and contains fat. We insist on the importance of the sonication of DPPH prior to use to obtain a good solubility of the product. We added a complementary centrifugation to get rid of the interferences caused by fat, and on the measurement of absorbance at 517 nm with a nanodrop, so that the volume required is importantly reduced. This technique could be used for the following purpose: antioxidant capacity assessment in small samples such breast milk and colostrum, but it could also be applied to the industry to assess the antioxidant capacity of dairy products, using reduced volumes. Finally, for medical use, this technique could be adopted to assess antioxidant capacity in body fluids which sample volume is very limited, such as tears for example, which determination was impossible with the previous method.